MX2011003238A - IDENTIFICATION AND USE OF BACTERIAL [2Fe-2S] DIHYDROXY-ACID DEHYDRATASES. - Google Patents

Abstract

A group of bacterial dihydroxy-acid dehydratases having a [2Fe-2S] cluster was discovered. Bacterial [2Fe-2S] DHADs were expressed as heterologous proteins in bacteria and yeast cells, providing DHAD activity for conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. Isobutanol and other compounds may be synthesized in pathways that include bacterial [2Fe-2S] DHAD activity.

Description

IDENTIFICATION AND USE OF DIHYDROXYACYD DEHYDRATASES [2Fe-2S] BACTERIAL FIELD OF THE INVENTION The invention relates to the field of industrial microbiology and to the expression of dihydroxy acid dehydratase activity. More specifically, to bacterial dihydroxy acid dehydratases with a [2Fe-2S] group that are identified and expressed as heterologous proteins in bacterial and yeast hosts.

BACKGROUND OF THE INVENTION Dihydroxy acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate and 2,3-dihydroxymethylvalerate to a-ketomethylvalerate. The DHAD enzyme, classified as E.C. 4.2.1.9, is part of biosynthetic routes of natural origin that produce valine, isoleucine, leucine and pantothenic acid (vitamin B5). The increased expression of DHAD activity is convenient for improved microbial production of branched chain amino acids or pantothenic acid.

DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to -cetoisovalerate is also a common step in the multiple biosynthetic routes of isobutanol that Ref. -.218182 they are described in the publication of the jointly owned and co-pending patent application of the United States no. 20070092957 Al. It describes the genetic manipulation of recombinant microorganisms for the production of isobutanol. Isobutanol is useful as an additive for fuels, whose availability can reduce the demand for petrochemical fuels.

For an improved production of compounds synthesized in routes that include dihydroxy acid dehydratase, it is convenient to express a heterologous enzyme that provides this enzymatic activity in the production host of interest. Obtaining a high functional expression of dihydroxyacid dehydratases in a heterologous host is complicated because the enzyme requires a Fe-S group, which implies the availability and appropriate loading of the group on the apoprotein of DHAD.

DHAD enzymes that require Fe-S groups are known in the art and are in the form [4Fe-4S] or [2Fe-2S]. Some bacterial enzymes are known, and the best characterized of these is E. coli (Flint, DH, et al (1993) J. Biol. Chem. 268: 14732-14742). However, all these bacterial enzymes are in the form [4Fe-4S]. The only form [2Fe-2S] reported to date is an enzyme from spinach (Flint and Emptage (1988) J. Biol. Chem. 263: 3558-3564).

It is convenient to use the [2Fe-2S] form of the enzyme in host cells to improve the production of routes introduced biosynthetics, since the form [2Fe-2S] creates a lower charge in the assembly and / or synthesis of the Fe-S group. Unfortunately, only one form [2Fe-2S] of this enzyme is known.

Therefore, there is a need to identify new forms [2Fe-2S] of DHAD for use in recombinant host cells, where the conversion of 2,3-dihydroxyisovalerate to -cetoisovalerate is a stage of the metabolic pathway in a biosynthetic pathway desired.

BRIEF DESCRIPTION OF THE INVENTION A method for identifying DHAD enzymes [2Fe-2S]; The method includes: a) search in a database one or more amino acid sequences with a profile of hidden Markov models prepared using the secs proteins. with no. ID: 164, 168, 230, 232, 298, 310, 344 and 346, wherein a match with an E value less than 10"5 provides a first subset of sequences, whereby the first subset of sequences corresponds to one or more proteins related to DHAD; b) analyzing the first subset of sequences corresponding to one or more DHAD-related proteins of step (a) to determine the presence of three conserved cysteines corresponding to positions 56, 129 and 201 in the amino acid sequence of the dihydroxyacid dehydratase (SEQ ID: 168) of Streptococcus mutans, whereby a second subset of sequences encoding DHAD enzymes [2Fe-2S] is identified; Y c) analyzing the second subset of sequences from step (b) to determine the presence of distinctive conserved amino acids at the positions corresponding to the positions in the amino acid sequence of the DHAD (sec. with ident. no .: 168) of Streptococcus mutans that are aspartic acid in position 88, arginine or asparagine in position 142, asparagine in position 208, and leucine in position 454, by virtue of which a third subset of sequences encoding DHAD enzymes is also identified [2Fe -2S].

In another aspect of the invention, the aforementioned method comprises, in addition: d) expressing a polypeptide having a sequence identifiable by any or all of steps a), b) and c) in a cell; Y e) confirming that the polypeptide has DHAD activity in the cell.

In another aspect of the invention the aforementioned method comprises, in addition: d) purify a protein encoded by a sequence identifiable by any or all of steps a), b) and e); Y e) confirm that the protein is a DHAD enzyme [2Fe-2S] by visible ultraviolet spectroscopy (UV vis) and EPR (electronic paramagnetic resonance, for its acronym in English).

In another aspect of the invention the aforementioned method further comprises selecting one or more of the sequences corresponding to the sequences of the bacterial DHAD [2Fe-2S] enzyme identified in any or all of steps a), b ) and e). The selected sequences can be expressed in a cell; and the DHAD activity in the cell can be confirmed. The selected sequences can be further purified to obtain a purified protein, and the activity of the DHAD enzyme [2Fe-2S] of that purified protein can be confirmed by UV vis spectroscopy and EPR spectroscopy.

Another aspect of the invention is directed to a microbial host cell comprising at least one heterologous DHAD [2Fe-2S] enzyme that can be identified with the methods described in the present disclosure. The cell can be a bacterial cell or a yeast cell. The cell can also be a recombinant cell that produces isobutanol.

Another aspect of the invention is a method for the production of isobutanol; The method includes: a) providing a microbial host cell comprising at least one heterologous DHAD [2Fe-2S] enzyme that can be identified with the methods described in the present disclosure, wherein the host cell further comprises a biosynthetic route of isobutanol; Y b) culturing the host cell of step (a) under conditions in which isobutanol is produced.

Another aspect of the invention is a method for the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate; The method includes: a) providing a microbial host cell comprising at least one heterologous DHAD [2Fe-2S] enzyme that can be identified with the methods described in the present disclosure and a source of 2,3-dihydroxyisovalerate; Y b) culturing the microbial host cell of (a) under conditions wherein 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate.

BRIEF DESCRIPTION OF THE FIGURES The present invention will be understood more easily from the following detailed description, the figures and descriptions of accompanying sequences, which form part of this application.

Figure 1 shows the regions of cisterns preserved, with C for bold cysteine, in a DHAD [4Fe-4S] and in representative DHAD [2Fe-2S] bacterial. Single-letter abbreviations are used for amino acids.

Figure 2 shows a phylogenetic tree of the proteins related to DHAD. The branches are labeled for DHAD [2Fe-2S] and [4Fe-4S], as well as the EDD, dehydratases of aldonic acid and an undefined group (Und). The selected DHADs are individually marked.

Figure 3 shows the biosynthetic routes for the production of isobutanol.

Figures 4A and 4B show stability charts of the activity in the presence of air for the DHAD of (Figure 4A) S. mutans, and (Figure 4B) DHAD of E. coli.

Figure 5 shows a tracing of the visible ultraviolet spectrum of the DHAD of S. mutans.

Figure 6 shows a plot of the electronic paramagnetic resonance spectrum (EPR) of the DHAD of S. mutans at different temperatures, between -253 ° C (20 ° K) and -183 ° C (90 ° K).

Figure 7 shows an HPLC analysis (high-performance liquid chromatography) of an extract of yeast cells expressing acetolactate synthase, KARI and DHAD genes from S. mutans, with a peak of isobutanol at 47,533 minutes Figure 8 shows a graph of stability of the DHAD of L. lactis in the presence of air.

Figure 9 shows a plot of the UV vis spectrum of DHAD of purified L. lactis.

Table 1 is a table of the HMM profile (Hidden Markov Model) for enzyme-based dihydroxy acid dehydratases with function tested according to the preparation described in Example 1. Table 1 is presented as an adjunct in the form electronics and is incorporated in the present description as a reference.

BRIEF DESCRIPTION OF THE SEQUENCES The following sequences comply with Title 37 of the C.F.R., 1821-1825 ("Requirements for patent applications containing descriptions of nucleotide sequences and / or amino acid sequences - sequences rules") according to the ST standard. 25 (1998) of the World Intellectual Property Organization (WIPO) and the requirements for EPO and PCT sequence listings (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for the nucleotide and amino acid sequence data comply with the rules described in Title 37 of C.F.R., §1.822.

Table 2a. Representative DHAD [2Fe-2S] bacterial proteins and coding sequences Methylobacterium nodulans ORS 2060 49 50 Rhodopseudomonas palustris BisB5 51 52 Rhodopseudomonas palustris BisB18 53 54 Bradyrhizobium sp. ORS278 55 56 Bradyrhizobium japonicum USDA 110 57 58 Fulvimarina pelagi HTCC2506 59 60 Aurantimonas sp. SI85-9A1 61 62 Hoeflea phototrophica DFL-43 63 64 Mesorhizobium loti MAFF303099 '65 66 Mesorhizobium sp. BNC1 67 68 Parvibaculum lavamentivorans DS-1 69 70 Loktanella vestfoldensis SKA53 71 72 Roseobacter sp. CCS2 73 74 Dinoroseobacter shibae DFL 12 75 76 Roseovarius nubinhibens ISM 77 78 Sagittula stellata E-37 79 80 Roseobacter sp. AzwK-3b 81 82 Roseovarius sp. TM1035 83 84 Oceanicola batsensis HTCC2597 85 86 Oceanicola granulosus HTCC2516 87 88 Rhodobacterales bacterium HTCC2150 89 90 Paracoccus denitrificans PD1222 91 92 Oceanibulbus indolifex HEL-45 93 94 Sulfitobacter sp. EE-36 95 96 Gamma proteobacterium marine not 181 182 cultivated EBAC20E09 Gamma proteobacterium not cultivated 183 184 eBACHOT4E07 Alcanivorax borkumensis SK2 185 186 Chromohalobacter salexigens DSM 3043 187 188 Marinobacter algicola DG893 189 190 Marinobacter aquaeolei VT8 191 192 Marinobacter sp. ELB17 193 194 Pseudoalteromonas haloplanktis TAC125 195 196 Acinetobacter sp. ADP1 197 198 Opitutaceae bacterium TAV2 199 200 Flavobacterium sp. MED217 201 202 Cellulophaga sp. MED134 203 204 Kordia algicida OT-1 205 206 Flavobacteriales bacterium ALC-1 207 208 Psychroflexus torquis ATCC 700755 209 210 Flavobacteriales bacterium HTCC2170 211 212 Unidentified Eubacterium SCB49 213 214 Gramella forsetii KT0803 215 216 Robiginitalea biformata HTCC2501 217 218 Tenacibaculum sp. MED152 219 220 Polaribacter irgensii 23 -P 221 222 Pedobacter sp. BAL39 223 224 Nitrosospira multiformis ATCC 25196 311 312 Chloroflexus aggregates DSM 9485 313 314 Leptospirillum sp. Group II UBA 315 316 Leptospirillum sp. Group II UBA 317 318 Halorhodospira halophila SL1 319 320 Nitrococcus mobilis Nb-231 321 322 Alkalilimnicola ehrlichei MLHE-1 323 324 Deinococcus geothermalis DSM 11300 325 326 Polynucleobacter sp. QLW-P1DMWA-1 327 328 Polynucleobacter necessarius STIR1 329 330 Azoarcus s. EbNl 331 332 Burkholderia phymatum STM815 333 334 Burkholderia xenovorans LB400 335 336 Burkholderia multivorans ATCC 17616 337 338 Burkholderia cenocepacia PC184 339 340 Burkholderia mallei GB8 horse 4 341 342 Ralstonia eutropha JMP134 343 344 Ralstonia metallidurans CH34 345 346 Ralstonia solanacearum UW551 347 348 Ralstonia pickettii 12J 349 350 Limnobacter sp. MEDI05 351 352 Herminiimonas arsenicoxydans 353 354 Bordetella parapertussis 355 356 Bordetella petrii DSM 12804 357 358 Polaromonas sp. JS666 359 360 Polaromonas naphthalenivorans CJ2 361 362 Rhodoferax ferrireducens T118 363 364 Verminephrobacter eiseniae EFOl-2 365 366 Acidovorax sp. JS42 367 368 Delftia acidovorans SPH-1 369 370 Methylibium petroleiphilum PM1 371 372 Gamma proteobacterium KT 71 373 374 Tremblaya princeps 375 376 Blastopirellula marina DSM 3645 377 378 Planctomyces maris DSM 8797 379 380 Salinibacter ruber DSM 13855 387 388 Table 2b. Other Representative DHAD [2Fe-2S] Bacterial Proteins and Coding Sequences Rhodobacterales bacterium HTCC2083 585 586 Candidatus Pelagibacter sp. HTCC7211 587 588 Chitinophaga pinensis DSM 2588 589 590 Alcanivorax sp. DG881 591 592 Micrococcus luteus NCTC 2665 593 594 Verrucomicrobiae bacterium DG1235 595 596 Synechococcus sp. PCC 7335 597 598 Brevundimonas sp. BAL3 599 600 Dyadobacter fermentans DSM 18053 601 602 Gamma proteobacterium NOR5-3 603 604 Gamma proteobacterium NOR51-B 605 606 Cyanobium sp. PCC 7001 607 608 Jonesia denitrificans DSM 20603 609 610 Brachybacterium faecium DSM 4810 611 612 Paenibacillus sp. JDR-2 613 614 Octadecabacter antarcticus 307 615 616 Variovorax paradoxus SI10 617 618 Table 3. Sequence identification numbers of other proteins and coding sequences The secs. with numbers ID: 395-409, 412-421, 424 and 431-436 are primers for PCR, sequencing or cloning analysis used and described in the examples of the present invention.

The sec. with no. Ident .: 410 is the nucleotide sequence of the pDMl vector.

The sec. with no. of ident. : 411 is the nucleotide sequence of the vector pLH532.

The sec. with no. Ident .: 425 is the FBA promoter of S. cerevisiae.

The sec. with no. Ident .: 430 is the nucleotide sequence of vector pRS423 FBA ilvD (Strep).

The sec. with no. Ident .: 437 is the nucleotide sequence of vector pNY13 The sec. with no. Ident .: 438 is the coding region of alsS from B. subtilis.

The sec. with no. Ident .: 439 is the GPD promoter of S. cerevisiae.

The sec. with no. Ident .: 440 is the CYC1 terminator of S. cerevisiae.

The sec. with no. of ident. : 442 is the ILV5 gene of S. cerevisiae.

DETAILED DESCRIPTION OF THE INVENTION As described in the present description, the applicants have solved the aforementioned problem through the discovery of methods to identify DHAD [2Fe-2S]. Through the discovery of these enzymes and their use in recombinant hosts, an activity advantage not previously appreciated in the genetic manipulation of routes with DHAD has been identified.

The present invention relates to bacterial or yeast recombinant cells engineered to provide the heterologous expression of dihydroxyacid dehydratases (DHAD) having a [2Fe-2S] group. The expressed DHAD acts as a component of a biosynthetic pathway for the production of a compound, such as valine, isoleucine, leucine, pantothenic acid or isobutanol. These amino acids and pantothenic acid can be used as nutritional supplements, and isobutanol can be used as an additive for fuels to reduce the demand for petrochemicals.

The following abbreviations and definitions will be used for the interpretation of the specification and the claims.

As used in the present description, the terms "comprising", "comprising", "including", "including", "having", "having", "containing" or "containing", or any other variant of these, they intend to cover a non-exclusive inclusion. For example, a composition, a mixture, a process, method, article or apparatus comprising a list of elements is not necessarily limited only to those elements, but may include others that are not expressly listed or are inherent in such a composition, mixture , process, method, article or device. In addition, unless expressly specified otherwise, the disjunction is related to an "or" inclusive and not with an "or" excluding. For example, a condition A or B is satisfied by any of the following criteria: A is true (or current) and B is false (or not current), A is false (or not current) and B is true (or current), and both A how B are true (or current).

Likewise, the indefinite articles "un (a)" and "ones" that precede an element or component of the invention are intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of the element or component. Therefore, "a" or "ones" must be construed to include one or at least one, and the singular form of the word of the element or component also includes the plural, unless the number obviously indicates which is unique The term "invention" or "present invention", as used in the present description, is a non-limiting term and is not intended to refer to any particular embodiment of the invention in particular, but encompasses all possible modalities as described in the specification and in the claims.

As used in the present description, the term "approximately", which modifies the amount of an ingredient or reagent used in the invention, refers to the variation that may occur in the numerical amount, for example, through liquid handling procedures and typical measurements used to prepare concentrates or real-world use solutions; through inadvertent errors in these procedures; through differences in the manufacture, origin or purity of the ingredients used to prepare the compositions or carry out the methods; and similar. The term "approximately" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a specific initial mixture. Whether or not modified by the term "approximately", the claims include equivalents for the quantities. In one embodiment the term "approximately" means an amount within 10% of the numerical value reported, preferably, within 5% of the numerical value reported.

The term "DHAD [2Fe-2S]" refers to DHAD enzymes that have a [2Fe-2S] group attached.

The term "DHAD [4Fe-4S]" refers to DHAD enzymes that have a group [4Fe-4S] bound. The term "dihydroxy acid dehydratase" is abbreviated DHAD and refers to an enzyme that converts 2,3-dihydroxyisovalerate to a-ketoisovalerate.

The term "biosynthetic route of isobutanol" refers to an enzymatic route to produce isobutanol from pyruvate.

The phrase "an facultative anaerobe" refers to a microorganism that can grow in both aerobic environments as anaerobes.

The phrases "carbon substrate" or "fermentable carbon substrate" refer to a carbon source capable of being metabolized by host organisms of the present invention and, specifically, to carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and single carbon substrates, or mixtures of these. Carbon substrates can include sugars of C6 and C5 and mixtures of these.

The term "gene" refers to a fragment of nucleic acid that is capable of being expressed as a specific protein that optionally includes the regulatory sequences that precede (5 'non-coding sequences) and that follow (3' non-coding sequences) to the coding sequence. "Native gene" refers to a gene as it is found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not native, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different origins, or regulatory sequences and coding sequences that are derived from the same origin, but are arranged in a different way from that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of a organism. A "foreign" or "heterologous" gene refers to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by means of a transformation procedure.

As used in the present description, the term "coding region" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refers to nucleotide sequences located upstream (5 'non-coding sequences), in or downstream (3' non-coding sequences) of a coding sequence and which influence transcription, processing or stability of the RNA or the translation of the associated coding sequence. Regulatory sequences may include promoters, leader translation sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site, and stem-loop structure.

The term "promoter" refers to a DNA sequence capable of controlling the expression of a functional RNA or coding sequence. Generally, a coding sequence is located in the 3 'direction with respect to a promoter sequence. The promoters can derive in their all of a native gene or may be composed of different elements derived from different promoters that are found in nature, or even comprise synthetic segments of DNA. Those skilled in the art will understand that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types most of the time are commonly referred to as "constitutive promoters". It is also recognized that since in most cases the exact boundaries of the regulatory sequences have not been fully defined, the DNA fragments of different lengths may have identical promoter activity.

The term "operably linked" refers to the association of nucleic acid sequences in a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). The coding sequences can be operatively linked to regulatory sequences in coding or non-coding orientation.

As used in the present description, the term "expression" refers to the transcription and stable accumulation of coding RNA (ARm) or non-coding RNA derived from the nucleic acid fragment of the invention. The term may also refer to the translation of the mRNA into a polypeptide.

As used in the present description, the term "transformation" refers to the transfer of a nucleic acid fragment to a host organism, which produces a genetically stable inheritance. Host organisms that contain the transformed nucleic acid fragments are termed "transgenic", "recombinant" or "transformed" organisms.

The terms "plasmid" and "vector", as used in the present description, refer to an extrachromosomal element that frequently presents genes that are not part of the cell's central metabolism and, usually, are in the form of DNA molecules. circular double-stranded These elements can be autonomously replicating sequences, integrating sequences of genomes, nucleotide or phage sequences, linear or circular, of a single or double stranded RNA or DNA, derived from any source, where several of the nucleotide sequences have been linked or recombined into a single construct capable of introducing a fragment of a promoter and DNA sequence for a selected gene product together with the appropriate 3 'untranslated sequence in a cell.

As used in the present disclosure, the term "codon degeneracy" refers to the nature of the genetic code that allows variation of the nucleotide sequence without affecting the amino acid sequence of a coded polypeptide. The skilled artisan knows perfectly the "codon preference" exhibited by a specific host cell in the use of nucleotide codons to specify a given amino acid. Therefore, when a gene for improved expression is synthesized in a host cell, it is convenient to design the gene so that its frequency of codon usage approximates the preferred codon usage frequency of the host cell.

The expression "optimized by codons", in what refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical use of codons of the host organism without altering the polypeptide encoded by the DNA.

As used in the present description, the phrases "isolated nucleic acid fragment" or "isolated nucleic acid molecule" are used interchangeably and refer to a single or double stranded RNA or DNA polymer optionally containing synthetic non-nucleotide bases. natural or altered An isolated fragment of nucleic acid in the form of a DNA polymer can comprise one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is "hybridizable" with another nucleic acid fragment, such as cDNA, genomic DNA or RNA molecule, when a single-stranded form of the nucleic acid fragment can be paired with the other nucleic acid fragment under the appropriate conditions of temperature and ionic power of the solution. Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2 °. ed. , Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly, Chapter 11 and Table 11.1 of this manual (which are incorporated herein by reference). The conditions of temperature and ionic power determine the "stringency" of the hybridization. Stringency conditions can be adjusted to identify moderately similar fragments (such as homologous sequences from unrelated organisms) with very similar fragments (such as genes that duplicate functional enzymes of closely related organisms). The post-hybridization washes determine the stringency conditions. A set of preferred conditions uses a series of washes that start with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45 ° C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50 ° C for 30 min. A more stringent set of stringency conditions uses higher temperatures in which the washes are identical to those mentioned above, except that the temperature of the two final 30 minute washes in 0.2X SSC, 0.5% SDS was increased to 60 °. C. Another preferred set of highly stringent conditions uses two final washes in 0. IX SSC, 0.1% SDS at 65 ° C. Another set of stringent conditions includes hybridization at 0.1X SSC, 0.1% SDS, 65 ° C and washes with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between the two nucleotide sequences, the higher the Tm value for nucleic acid hybrids having these sequences. The relative stability (corresponding to a higher value of Tm) of nucleic acid hybridizations decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids of more than 100 nucleotides of length, equations have been derived to calculate Tm (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, ie, oligonucleotides, the position of the mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8). In one embodiment, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, the minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably, at least about 20 nucleotides; and, most preferably, the length is at least about 30 nucleotides. In addition, the skilled artisan will recognize that the temperature and salt concentration of the wash solution may be regulated as necessary in accordance with factors such as the length of the probe.

A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising a sufficient amount of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by a person skilled in the art or by comparison and computer-controlled sequence identification by the use of algorithms, such as BLAST (Altschul, S.F., et al., J. Mol. Biol., 215: 403-410 (1993)). Generally, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is needed to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. In addition, with respect to the nucleotide sequences, oligonucleotide probes specific for genes comprising 20 to 30 contiguous nucleotides can be used in sequence dependent methods for identification (eg, Southern hybridization) and isolation (eg, hybridization if your of bacterial colonies or bacteriophage plaques) of genes. In addition, short 12-15 base oligonucleotides can be used as primers for PCR amplification in order to obtain a specific nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises a sufficient amount of the sequence to specifically identify and / or isolate a nucleic acid fragment comprising the sequence. The specification of the present instructs on the complete sequence of amino acids and nucleotides that encode specific proteins. The experienced technician, with the benefit of the sequences as reported in the present description, can now use all or a substantial portion of the described sequences for purposes known to those skilled in the art. Therefore, the present invention comprises the complete sequences as reported in the attached Sequence Listing, as well as the substantial portions of those sequences, as defined above.

The term "complementary" is used to describe the relationship between nucleotide bases that are capable of interacting with each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine.

The phrase "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" or "sequence identity" also means the degree of sequence relationship between the polypeptide or polynucleotide sequences, as the case may be, as determined by the sequence matching of these sequences. "Identity" and "similarity" can be readily calculated by known methods including, but not limited to, those described in: 1.) Computational Molecular Biology (Lesk, A.M., Ed.) Oxford University: NY (1988) ); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

The preferred methods for determining identity are designed to give the best match between the sequences under test. The methods for determining identity and similarity are codified in publicly available software. The sequence alignment calculations and identity percentages can be performed with the MegAlign ™ program of the LASERGENE bioinformatics program suite (DNASTAR Inc., Madison, WI). The multiple alignment of the sequences is done with the "Clustal Alignment Method", which covers different varieties of the algorithm that include the "Clustal V alignment method" that corresponds to the alignment method identified as Clustal V (described by Higgins and Sharp, CABIOS 5: 151-153 (1989), Higgins, DG et al., Coput Appl. Biosci., 8: 189-191 (1992)) and included in the MegAlign ™ program of the bioinformatics program LASERGENE (DNASTAR Inc. .). For multiple alignments, the default values correspond to PENALTY OF INTERRUPTION = 10 and PENALTY OF INTERRUPTION LENGTH10. The default parameters for the pairwise alignments and for the calculation of the percent identity of protein sequences with the Clustal method are KTUPLE = 1, PENALTY OF INTERRUPTION3, WINDOW = 5 and DIAGONALS SAVED = 5. For acids nucleic, these parameters are KTUPLE = 2, PENALTY OF INTERRUPTION = 5, WINDOW = 4 and DIAGONALS SAVED = 4. After aligning the sequences with the Clustal V program, it is possible to obtain a "percent identity" by observing the "sequence distances" table in the same program. In addition, the "Clustal W alignment method" is available corresponding to the alignment method identified as Clustal W (described by Higgins and Sharp, CABIOS 5: 151-153 (1989), Higgins, DG et al., Comput. Biosci 8: 189-191 (1992) Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc Acid Res. 22: 4673 4680) and included in the program MegAlign ™ v6.1 of the program package bioinformatics of LASERGENE (DNASTAR Inc.). Default parameters for multiple alignment (PENALTY OF GAP = 10, PENALTY BY LENGTH OF GAP = 0.2, Delay of divergent sequences (%) = 30, DNA transition weight = 0.5, weight matrix for proteins = Gonnet series, matrix of weights for the DNA = IUB). After the alignment of the sequences through the use of the Clustal program, it is possible to obtain a "percentage of identity" when viewing the table of "sequence distances" in the same program.

A person skilled in the art will very well understand that to identify polypeptides from other species, wherein the polypeptides have the same or similar function or activity, various levels of protein are useful. sequence identity. Useful examples of identities percentages include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any entire percentage of 55% to 100% to describe the present invention, such as 55%, 56%, 57%, 58 59 60%, 61 62%, 63%, 64%, 65 66%, 67%, 68%, 69%, 70%, 71 72%, 73, 74%, 75%, 76%, 77%, 78 79 80%, 81%, 82%, 83%, 84%, 85 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 The appropriate nucleic acid fragments not only have the homologies mentioned above, but typically encode a polypeptide that has at least 50 amino acids, preferably, at least 100 amino acids, more preferably, at least 150 amino acids, even more preferably, at least 200 amino acids and, most preferably, at least 250 amino acids.

The phrase "software / sequence analysis program" refers to any computational algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. The "software / sequence analysis program" may be commercially available or developed independently. Typical sequence analysis software includes, but is not limited to: 1.) the set of GCG programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215: 403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. adison, WI); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, MI); and 5.) the FASTA program that incorporates the Smith-Waterman algorithm (WR Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting date, 1992, 111-20. ): Suhai, Sandor, Plenum: New York, NY). Within the context of this application, it will be understood that when the analysis is performed with the sequence analysis program, the results of the analysis will be based on the "predetermined values" of the reference program, unless it is specified in any other way. As used in the present description, "predetermined values" refers to any set of values or parameters that are originally loaded with the program the first time it is started.

The standard techniques of molecular cloning and recombinant DNA used in the present description are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter, "Maniatis"); and in Silhavy, T.J., Bennan, M.L. and Enquist, L.W., Experiment with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Other methods used here are in Methods in Enzymology, Volume 194, Guiae to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, CA ).

Discovery of DHAD [2Fe-2S] The DHAD proteins are known to contain an iron-sulfur linked group (Fe-S) that is required for enzymatic activity. The only DHAD with a [2Fe-2S] group reported to date is a spinach enzyme (Flint and Emptage (1988) J. Biol. Chem. 263: 3558-3564). Some bacterial enzymes are also known, and the best characterized of these is E. coli (Flint, DH, et al (1993) J. Biol. Chem. 268: 14732-14742), which has a group [4Fe-4S ] Applicants have now determined that there is a class of bacterial DHADs that contain a [2Fe-2S] group (the DHAD [2Fe-2S]). Applicants have discovered that the DHAD group [2Fe-2S] can be distinguished from a group of DHAD [4Fe-4S] by the presence of three conserved cysteine residues in the protein. The three conserved cysteines are similar to the three essential cysteines described in arabonato dehydratase (an aldonic acid) of Azospirillum brasiliense (Watanabe, S et al., J. Biol. Chem. (2006) 281: 33521-33536) which is reported to contain a group [4Fe-4S]. In the protein arabonato dehidratasa of Azospirillum brasiliense, determined that the cysteines located at the positions of amino acids 56, 124 and 197 were essential for the enzymatic activity and probably participated in coordination with the Fe-S group. Surprisingly, the applicants have discovered that three conserved cysteines that are in the positions corresponding to the three essential cysteines of the Arabido dehydratase of Azospirillum brasiliense are characteristic of DHAD containing a [2Fe-2S] group. Applicants have discovered that DHAD from E. coli, which contains a group [4Fe-4S], has two of the conserved cysteines, but not the third conserved cysteine. Figure 1 shows a comparison of the regions of the conserved cysteine sequences for the DHAD of E. coli containing [4Fe-4S] groups and for representative cases of a phylogenetic group of the DHDA with groups [2Fe-2S] ] which was identified in the present in Example 1 and which is described below.

Applicants have developed a method to identify DHAD [2Fe-2S]. In the present invention bacterial DHAD [2Fe-2S], which can be identified with this method, can be used for heterologous expression in bacteria.

To structurally characterize the DHAD enzymes, a profile of hidden Markov models (HMM) was prepared, as described in Example 1, by the use of amino acid sequences of DHAD proteins with experimentally verified function, as determined in Example 2 herein, and are provided in Table 1. These DHADs are from Nitrosomonas europaea (DNA, sec. with ident. : 309; protein, sec. With ident. No .: 310), Synechocystis sp. PCC6803 (DNA, sec with ident. No .: 297; protein, sec. With ident. No .: 298), Streptococcus mutans (DNA, sec. With ident. No .: 167; protein, sec. Ident .: 168), Streptococcus thermophilus (DNA, sec. with ident. no .: 163; sec. with ident. no .: 164), Ralstonia metallidurans (DNA, sec. with ident. no .: 345; protein, sec. With ident. No .: 346), Ralstonia eutropha (DNA, sec. With ident. No .: 343; protein, sec. With ident. No .: 344), and Lactococcus lactis ( DNA, sec. With ident. No .: 231; protein, sec. With ident. No .: 232). In addition, it was found that the DHAD of Flavobacterium johnsoniae (DNA, sec.with ident .: 229; protein, sec.with ident .: 230) had dihydroxy acid dehydratase activity when expressed in E. coli and was used to elaborate the profile. This HMM profile for DHADs can be used to identify proteins related to DHAD. Any protein that matches the HMM profile that has an E value of < 10"5 is a DHAD-related protein, which includes DHAD [4Fe-4S], DHAD [2Fe-2S], dehydratases of aldonic acid and phosphogluconate dehydratases, a phylogenetic tree of sequences that match this profile HMM is shown in Figure 2.

Then, the sequences matching the HMM profile given here are analyzed to determine the presence of the three conserved cysteines described above. The exact positions of the three conserved cysteines can vary, and these can be identified in the context of the surrounding sequence by using multiple sequence alignments made with the Clustal W algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc Acid Res. 22: 4673 4680) by using the following parameters: 1) for parameters of pairwise alignment, an interruption aperture = 10; extension of the interruption = 0.1; the matrix is Gonnet 250; and the - Slow-exact mode, 2) for parameters of multiple alignment, interrupt opening = 10; extension of the interruption = 0.2; and the matrix is the Gonnet series. For example, the three conserved cysteines are located at the positions of amino acids 56, 129 and 201 in the DHAD of Streptococcus mutans (sec.with ident.ID: 168), and at amino acid positions 61, 135 and 207 in DHAD of Lactococcus lactis (sec. with ident. no .: 232). The exact positions of the three conserved cysteines in other protein sequences correspond to these positions in the amino acid sequence of S. mutans or L. lactis. A person skilled in the art will easily identify the presence or absence of each of the three cysteines conserved in the amino acid sequence of the DHAD protein when using pairwise or multiple sequence alignments. In addition, other methods can be used to determine the presence of the three conserved cysteines, such as a visual analysis.

Proteins matching the HMM profile for DHAD that have two conserved cysteines, but not the third (position 56) include the DHAD [4Fe-4S] and phosphogluconate dehydratases (EDD). Proteins that have all three conserved cysteines include arabonate dehydratases and DHAD [2Fe-2S], and are members of a DHAD [2Fe-2S] / aldonic acid dehydratases group. DHAD [2Fe-2S] can be distinguished from aldonic acid dehydratases by an analysis to determine the distinctive conserved amino acids that were found to be present in DHAD [2Fe-2S] or aldonic acid dehydratases at positions corresponding to following positions in the amino acid sequence of the DHAD of Streptococcus mutans. These distinctive amino acids are in DHAD [2Fe-2S] or aldonic acid dehydratases, respectively, in the following positions (with an appearance frequency greater than 90%): 88 asparagine compared to glutamic acid; 113 not preserved compared to glutamic acid; 142 arginine or asparagine compared to non-conserved; 165: not preserved compared to glycine; 208 asparagine compared to non-conserved; 454 leucine compared to non-conserved; 477 phenylalanine or tyrosine compared to non-conserved; and 487 glycine compared to not preserved.

The methods described for the identification of DHAD enzymes [2Fe-2S] can be carried out in a single sequence or in a group of sequences. In a preferred embodiment, one or more sequence databases with an HMM profile are queried, as described in the present description. Suitable sequence databases are known to those skilled in the art and include, but are not limited to, the database for non-redundant proteins Genbank, the SwissProt database, or the UniProt database or other bases of existing data, such as GQPat (GenomeQuest, estboro, MA) and BRENDA (Biobase, Beverly, MA).

Among the DHAD [2Fe-2S] that can be identified with this method, bacterial DHAD [2Fe-2S] can be easily identified through the organism of natural origin which is a type of bacteria. Any bacterial DHAD [2Fe-2S] that can be identified with this method may be suitable for heterologous expression in a microbial host cell. It will also be understood that any bacterial DHAD [2Fe-2S] described expressly in the present description per sequence may be suitable for heterologous expression in bacterial cells.

The preferred bacterial DHAD [2Fe-2S] enzymes can be expressed in a host cell and provide DHAD activity.

Initially, 193 different bacterial DHAD [2Fe-2S] were identified with sequence identities less than 95% (proteins with identity greater than 95% were deleted to simplify the analysis), as described in Example 1, and the sequences of amino acids and coding for these proteins are provided in the sequence listing; The sequence identification numbers are listed in Table 2a.

A subsequent analysis described in Example 11 produced 268 different bacterial DHAD [2Fe-2S]. The amino acid and coding sequences that were not identical to any of the bacterial DHAD [2Fe-2S] 193 provided by the initial identification are included in the sequence listing, and the sequence identification numbers are listed in Table 2b.

Any DHAD [2Fe-2S] protein that matches an identifiable sequence through the methods described in the present disclosure with a sequence expressly described in the present disclosure with an identity of at least about 95%, 96%, 97% , 98% or 99% is a DHAD [2Fe-2S] that can be used for heterologous expression in bacterial cells, as described in the present disclosure. Among the bacterial DHAD [2Fe-2S] expressly described in present invention, there is 100% conservation of distinctive amino acids in the positions: 88 aspartic acid, 142 arginine or asparagine, 208 asparagine and 454 leucine.

In addition, the bacterial DHAD [2Fe-2S] that can be used in the present invention are 'identifiable by their position in the DHAD branch [2Fe-2S] of a phylogenetic tree of DHAD-related proteins, such as the one shown. in Figure 2 and described in Example 1. In addition, the bacterial DHAD [2Fe-2S] that can be used are identifiable by using sequence comparisons with any of the bacterial DHARD [2Fe-2S] 281, whose sequences are provided in the present invention, wherein the sequence identity can be at least about 80% -85%, 85% -90%, 90% -95% or 95% -99%.

In addition, the sequences of the DHAD [2Fe-2S] provided in the present invention can be used to identify other homologs in nature. For example, each of the DHADs encoding nucleic acid fragments described in the present disclosure can be used to isolate genes encoding homologous proteins. The isolation of homologous genes by means of protocols that depend on sequences is well known in the art. Examples of protocols that depend on sequences include, but are not limited to: 1.) nucleic acid hybridization methods; 2.) DNA and RNA amplification methods, such as is illustrated by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Patent No. 4,683,202; the ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. United States 82: 1074 (1985); or chain shift amplification (SDA, for its acronym in English), Walker, et al., Proc. Nati Acad. Sci. United States, 89: 392 (1992)]; and 3.) methods of construction of libraries and complementation analysis.

For example, genes encoding proteins or polypeptides similar to DHAD [2Fe-2S] encoding genes provided in the present invention could be isolated directly by using all or a portion of the nucleic acid fragments of the invention as hybridization probes of DNA to select libraries from any desired organism by using methodology well known to those skilled in the art. Specific oligonucleotide probes based on the described nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, all sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (for example, DNA labeling techniques with random primers, nick translation or labeling). ends), or RNA probes by the use of existing in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part (or the total length) of the sequences of the present disclosure. The products resulting from the amplification can be labeled directly during the amplification reactions or after the amplification reactions, and can be used as probes to isolate full-length DNA fragments by hybridization under suitable stringency conditions.

Typically, in amplification techniques of the PCR type, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers must be designed to provide an efficient and faithful replication of the target nucleic acid. Primer design methods for PCR are common and well known in the art (Thein and Wallace, "The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, KE Davis Ed. ., (1986) pp. 33-50, IRL: Herndon, VA; and Rychlik,., In Methods in Molecular Biology, White, BA Ed., (1993) Vol. 15, pp. 31-39, PCR Protocols: Current Methods and Applications, Humania: Totowa, NJ).

Generally, two short segments of the described sequences can be used in reaction protocols in polymerase chain to amplify longer nucleic acid fragments that encode DNA or RNA homologous genes. The polymerase chain reaction can also be performed on a library of cloned nucleic acid fragments, wherein the sequence of a primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid to the 3 'end of the mRNA precursor encoding microbial genes.

Alternatively, the sequence of the second primer may be based on sequences derived from the cloning vector. For example, the experienced technician may follow the RACE protocol (Frohman et al., PNAS, United States 85: 8998 (1988)) to generate the cDNAs by using a PCR to amplify copies of the region between a single point in the transcript and the 3 'or 5' end. The primers oriented in the directions of the 3 'and 51 ends can be designed from the sequences of the present disclosure. By using commercially available RACE 3 'or RACE 5' systems (eg, BRL, Gaithersburg, MD), specific 3 'or 5' fragments of the cDNA can be isolated (Ohara et al., PNAS, United States 86: 5673 (1989); Loh et al., Science 243: 217 (1989)).

Alternatively, the provided coding sequences of DHAD [2Fe-2S] can be used as hybridization reagents for the identification of homologs.

The building blocks of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. The probes are,. typically, single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. The probes are "hybridizable" for the nucleic acid sequence that will be detected. The length of the probe can vary from 5 bases to tens of thousands of bases and will depend on the specific test that will be practiced. Typically, a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule needs to be complementary to the nucleic acid sequence that will be detected. In addition, it is not necessary that the complementarity between the probe and the target sequence is perfect. Hybridization will occur between the molecules of imperfect complementarity with the result that a certain fraction of the bases in the hybridized region will not be paired with the appropriate complementary base.

The hybridization methods are well defined.

Typically, the probe and the sample must be mixed under conditions that allow the hybridization of the nucleic acids. This involves contacting the probe and the sample in the presence of an organic or inorganic salt at the appropriate concentration and temperature conditions. The probe and the nucleic acids of the sample must be in contact for a sufficiently long time so that any possible hybridization between the probe and the nucleic acids of the sample can occur. The concentration of the probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the concentration of the probe or target, the shorter the time it will take for the incubation of the hybridization. Optionally, a chaotropic agent can be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. In addition, the chaotropic agent allows for the sensitive and rigorous hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucí Acids Res. 19: 5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, formamide can be added to the hybridization mixture, typically, 30 to 50% (v / v).

Various hybridization solutions can be employed. Typically, they comprise from about 20 to 60% by volume, preferably 30%, of an organic polar solvent.

A common hybridization solution employs about 30 to 50% v / v of formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffer solution (eg, sodium citrate, Tris-HCl, PIPES or HEPES (pH range of about 6-9)), from about 0.05 to 0.2% detergent (eg, sodium dodecyl sulfate), or from 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (approximately 300 -500 kdal), polyvinyl pyrrolidone (approximately 250-500 kdal) and serum albumin. Also included in the standard hybridization solution are unlabeled carrier nucleic acids of about 0.1 to 5 mg / ml, fragmented nucleic acid, (eg, calf thymus or salmon sperm DNA, or yeast RNA) and, optionally, , from about 0.5 to 2% weight / vol. of glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar or water-soluble polar agents, (eg, polyethylene glycol), anionic polymers (eg, polyacrylate or polymethylacrylate), and anionic saccharide polymers, ( for example, dextran sulfate).

Nucleic acid hybridization can be adapted to a variety of assay formats. One of the most suitable is the interleaved test format. Specifically, the sandwich assay can be adapted to hybridization under non-denaturing conditions. A primary component of a test of Interleaved type is a solid support. The solid support has adsorbed or covalently coupled to it a non-labeled immobilized nucleic acid probe and which is complementary to a portion of the sequence.

Expression of heterologous bacterial DHAD [2Fe-2S] in bacterial and yeast hosts Applicants have discovered that a heterologous DHAD [2Fe-2S] provides DHAD activity when expressed in a microbial cell. Any DHAD [2Fe-2S] that can be identified as described herein can be expressed in a heterologous microbial cell. Expression of any of these proteins provides DHAD activity for a biosynthetic pathway that includes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to -cetyletylvalerate. The expression of a DHAD [2Fe-2S], as opposed to a DHAD 4Fe-4D, reduces the requirement of Fe and S groups to obtain enzymatic activity. In addition, it was found in the present invention that DHAD [2Fe-2S] of S. mutans has greater stability in the presence of air compared to the sensitivity in the presence of DHAD air [4Fe-4S] of E. coli, which is desirable to obtain a better activity in a heterologous host cell.

Bacterial cells that may also be hosts for expression of a heterologous bacterial DHAD [2Fe-2S] include, but are not limited to, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. Manipulating gene expression of a heterologous bacterial DHAD [2Fe-2S] can increase DHAD activity in a bacterial host cell that naturally expresses a DHAD [2Fe-2S] or a DHAD [4Fe-4S]. These host cells may include, for example, E. coli and Bacillus subtilis. In addition, genetically manipulating the expression of a heterologous bacterial DHAD [2Fe-2S] DHAD provides DHAD activity in a bacterial host cell that has no endogenous DHAD activity. These host cells may include, for example, Lactobacillus, Enterococcus, Pediococcus and Leuconostoc.

Specific hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus lichenifor is, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinariu, Enterococcus faecalis and Bacillus subtilis.

A bacterial host cell can be genetically engineered to express a heterologous bacterial DHAD [2Fe-2S] by methods well known to a person skilled in the art. The coding region of DHAD that will be expressed can be optimized by codons for the target host cell, as is well known to a person skilled in the art. Vectors useful for the transformation of a variety of host cells are common and are commercially available through companies such as EPICENTRE® (Madison, WI), Invitrogen Corp. (Carlsbad, CA), Stratagene (La Jolla, CA), and New England Biolabs, Inc. (Beverly, MA). Typically, the vector contains a selectable marker and sequences that allow for autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors can comprise a promoter region harboring transcriptional initiation controls and a transcriptional termination control region between which a fragment of DNA coding region can be inserted to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it should be understood that these control regions can also be derived from genes that are not native to the specific species selected as the production host.

Promoters or initiation control regions that are useful for driving the expression of the bacterial DHAD [2Fe-2S] coding regions in the desired host cell are numerous and known to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention, which include, but are not limited to, lac, ara, tet, trp, 1PL, lPRl T7, tac and trc promoters (useful for expression in Escherichia coli, Alcaligenes and Pseudomonas); amy, apr and npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis and Paenibacillus acerans; nisA (useful for expression in gram-positive bacteria, Eichenbaum et al., Appl. Environ Microbiol. 64 (8): 2763-2769 (1998)); and the synthetic Pll promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152: 1011-1019 (2006)).

The termination control regions can also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, if it is included, it is most preferred.

Certain vectors have the ability to replicate in a wide range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors are available: pRK437, pRK442 and pRK442 (H). These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid 50 (l): 74-79 (2003)). Several plasmid derivatives of the broad host range plasmid RSF1010, IncP4, are also available, with promoters that can act in a range of Gram negative bacteria.

Plasmids pAYC36 and pAYC37 have active promoters together with multiple cloning sites to allow the expression of heterologous genes in Gram negative bacteria. Some vectors that are useful for the transformation of Bacillus subtilis and Lactobacillus include ??? ß? and derivatives thereof (Renault et al., Gene 183: 175-182 (1996); and O'Sullivan et al., Gene 137: 227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl. Environ Microbiol., 62: 1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol 184: 5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ Microbiol. 63: 4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ Microbiol. 67: 1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob, Agents Chemother, 38: 1899-1903 (1994)). Several Lactobacillus plantarum plasmids have also been described (van Kranenburg et al., Appl. Environ.Microbiol.71 (3): 1223-1230 (2005)).

In addition, there is a wide availability of chromosomal gene replacement tools. For example, a thermosensitive variant of the host wide range replicon pWVIO1 has been modified to construct a plasmid pVE6002 which can be used to effect the replacement of genes in a range of gram positive bacteria (Maguin et al., J. Bacteriol. 17): 5633-5638 (1992)). In addition, in vitro transposomes are available in the market, for example, EPICENTRE®, to create random mutations in a variety of genomes.

Yeast cells that can be hosts for the expression of a heterologous bacterial DHAD [2Fe-2S] are any yeast cells that can be subjected to genetic manipulation and include, but are not limited to, Saccharomyces, Schizosaccharo yces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis and Yarrowia lipolytica. The most suitable is Saccharomyces cerevisiae.

Expression is achieved by transformation with a gene comprising a sequence encoding any of these DHAD [2Fe-2S]. The coding region of DHAD that will be expressed can be optimized by codons for the target host cell, as is well known to a person skilled in the art. Methods for the expression of genes in yeast are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink ( Eds.), Elsevier Academic Press, San Diego, CA) Gene expression in yeast typically requires a promoter operably linked to a coding region of interest, and a transcriptional terminator.Several yeast promoters can be used to construct the cassettes of expression for genes in yeast, which include, but are not limited to, promoters derived from the following genes: CYC1, HIS3, GAL1, GALIO, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, GPM, and AQX1 . Suitable transcriptional terminators include, but are not limited to, FBAt, GPDt, GPMt, ERGIOt, GALlt, CYC1 and ADH1.

Suitable promoters, transcriptional terminators and DHAD [2Fe-2S] coding regions can be cloned into E. coli yeast shuttle vectors and transformed into yeast cells. These vectors allow the propagation of strains both in strains of E. coli and in strains of yeast. Typically, the vector used contains a selectable marker and sequences that allow for autonomous replication or chromosomal integration in the desired host. Typically, the plasmids used in yeast are the transporter vectors pRS423, pRS424, pRS425 and pRS426 (Collection of US-type cultures, ATCC, Rockville, MD), which contain an E. coli origin of replication ( for example, pMBl), a yeast replication origin of 2 μ and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trpl (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). The construction of expression vectors with a chimeric gene encoding the DHADs described can be performed by standard techniques of molecular cloning in E. coli or by the method of recombination by repair of the interruption in yeast.

The cloning method of interruption repair takes advantage of highly efficient homologous recombination in yeast. Typically, a DNA from a yeast vector is digested (eg, at its multiple cloning site) to create an interruption in its sequence. Several inserts of the DNA of interest containing a sequence of = 21 bp are generated at both 5 'and 3' ends, which are sequentially superimposed on each other., and with the 5 'and 3' terminal of the vector DNA. For example, to construct an expression vector in yeast for "Gen X", a yeast promoter and a yeast terminator are selected for expression cystein. The promoter and the terminator are amplified from the genomic DNA of the yeast, and the X gene is amplified either by PCR from its parent organism or obtained from a cloning vector comprising the Gen X sequence. at least there is a 21 bp overlap sequence between the 5 'end of the linearized vector and the promoter sequence, between the promoter and the X gene, between the X gene and the terminator sequence, and between the terminator and the 3' end of the linearized vector. Then, the interrupted vector and the DNA inserts are cotransformed into a yeast strain and plated in a plate in the medium containing the appropriate mixtures of compounds that allow the complementation of the nutritional selection markers in the plasmids. The presence of the correct combinations of inserts can be confirmed by PCR mapping using the plasmid DNA prepared from the selected cells. The isolated plasmid DNA of yeast (usually of low concentration) can then be transformed into an E. coli strain, eg, TOP10, followed by mini-preparations and restriction mapping to further verify the plasmid construct. Finally, the construct can be verified by sequence analysis.

Like the interruption repair technique, the integration into the yeast genome also takes advantage of the homologous recombination system in yeast. Typically, a cassette containing a coding region plus control elements (promoter and terminator) and an auxotrophic marker is amplified by PCR with a high fidelity DNA polymerase through the use of primers that hybridize with the cassette and contain from 40 to 70 base pairs of sequence homology for the 5 'and 3' regions of the genomic area where the insertion is desired. Then the PCR product is transformed into yeast and plated on a plate with a medium containing the appropriate mixtures of compounds that allow the selection of the integrated auxotrophic marker. For example, to integrate the "X gene" into the "Y" chromosomal location, the promoter-X-terminator coding region construct is amplified by PCR from a plasmid DNA construct and binds to an autotrophic marker (such as URA3) by means of SOE PCR (PCR with overlap extension binding method) or by common methods of cloning and restriction digestion. The complete cassette, containing the promoter-coding region X-terminator-region of URA3 is amplified by PCR with primer sequences containing from 40 to 70 bp of homology for the 5 'and 3' regions of the "Y" location in the chromosome of the yeast. The PCR product is transformed into yeast and selected in growth media without uracil. Transformants can be verified either by colony PCR or by direct chromosomal DNA sequencing.

Confirmation of DHAD activity The presence of DHAD activity in a genetically engineered cell to express a bacterial DHAD [2Fe-2S] can be confirmed by the use of methods known in the art. To give an example, and as demonstrated in the examples of the present invention, crude extracts from cells genetically engineered to express a bacterial DHAD [2Fe-2S] can be used in a DHAD assay, as described by Flint and Emptage (J. Biol. Chem. (1988) 263 (8): 3558-64) when using dinitrophenylhydrazine. In another example, as demonstrated in the examples of the present disclosure, DHAD activity can be evaluated by expressing a bacterial DHAD that can be identified with the methods described herein in a yeast strain that has no endogenous DHAD activity. If the DHAD activity is present, the yeast strain will grow in the absence of branched-chain amino acids. DHAD activity can also be confirmed with more indirect methods, such as by analyzing a product downstream of a route that requires DHAD activity. Any product having a-ketoisovalerate or -cetmethylvalerate can be measured as an intermediate of a route to assess DHAD activity. A list of these products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol and isobutanol.

Isobutanol production The expression of a bacterial DHAD [2Fe-2S] in bacteria or yeast, as described in the present disclosure, provides the recombinant host cell transformed with dihydroxy acid dehydratase activity for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2, 3-dihydroxymethylvalerate to -cetyletylvalerate. Any product having a-ketoisovalerate or a-ketomethylvalerate as the route intermediate can be most efficiently produced in the bacterial or yeast strains described in the present disclosure having the heterologous DHFR [2Fe-2S] described. A list of these products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol and isobutanol.

For example, valine biosynthesis in yeast includes the steps of conversion of acetolactate to 2,3-dihydroxy-isovalerate by acetohydroxy-acid reductoisomerase (ILV5), conversion of 2,3-dihydroxy-isovalerate to a-ketoisovalerate (also referred to as 2- ketoisovalerate) by the dihydroxy acid dehydratase, and conversion of α-ketoisovalerate to valine by branched chain amino acid transaminases (BAT2) and branched chain amino acid aminotransferases (BATI). Leucine biosynthesis includes the same steps for a-ketoisovalerate, followed by the conversion of α-ketoisovalerate to alpha-isopropyl malate by alpha-isopropyl malate synthase (LEU9, LEU4), conversion of alpha-isopropyl malate to beta-isopropyl malate by isopropyl maleate isomerase (LEU1), conversion of beta-isopropyl malate to alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2) and, finally, conversion of alpha-ketoisocaproate to leucine by transaminases of branched chain amino acids (BAT2) and amino acid aminotransferases of branched chain (BATI). The bacterial route is similar and involves proteins and genes named differently. The increased conversion of 2,3-dihydroxy-isovalerate to a-ketoisovalerate will increase the flow in these routes, particularly if one or more additional enzymes of a route are overexpressed. By for the production of valine or leucine, it is desired to use a strain described in the present invention.

The pantothenic acid biosynthesis includes a step carried out with DHAD, as well as the steps carried out with ketopantoate hydroxymethyltransferase and pantothenate synthase. The genetic manipulation of the expression of these enzymes for the improved production of pantothenic acid biosynthesis in microorganisms is described in U.S. Pat. 6,177,264.

The a-ketoisovalerate product of DHAD is an intermediate product in the biosynthetic routes of isobutanol described in the co-pending and jointly owned patent publication of the United States no. 20070092957 Al, which is incorporated in the present description as a reference. A diagram of the described biosynthetic routes of isobutanol is given in Figure 3. The production of isobutanol in a strain described in the present description benefits from increased DHAD activity. As described in the present disclosure, DHAD activity is provided by the expression of a bacterial DHAD [2Fe-2S] in a bacterial or yeast cell. As described in U.S. Patent No. 20070092957 Al, the steps in an example of the biosynthetic route of isobutanol include the conversion of: pyruvate to acetolactate (see Figure 1, step a of the route in that figure), as catalysed, for example, by acetolactate synthase, 2-, 3-dihydroxyisovalerate acetolactate (see Figure 1, step b of the route in that figure), as catalyzed, for example, by the acetohydroxy acid isomerreductase; 2,3-dihydroxyisovalerate to a-ketoisovalerate (see Figure 1, step c of the route in that figure), as catalyzed, for example, by the acetohydroxy acid dehydratase, also called dihydroxy acid dehydratase (DHAD); α-ketoisovalerate to isobutyraldehyde (see Figure 1, step d of the route in that figure), as catalyzed, for example, by the branched-chain acid decarboxylase; Y isobutyraldehyde to isobutanol (see Figure 1, step e of the route in that figure), as catalyzed, for example, by the branched-chain alcohol dehydrogenase.

The substrate for product conversions and the enzymes involved in these reactions for steps f, g, h, I, j, and k of alternative routes are described in U.S. Pat. 20070092957 Al.

The genes that can be used for expression of the enzymes of the aforementioned pathway steps that are not the bacterial DHAD [2Fe-2S] described in the present description, as well as those for two additional routes of isobutanol, are described in the patent of United States no. 20070092957 Al, and other genes that can be used can be identified by a person skilled in the art through bioinformatic or experimental methods, as described above. The preferred use in the three ketoacid reductaisomerase (KARI) enzyme pathways with particularly high activities is described in the co-pending and co-owned patent application publication of the United States no. 20080261230A1. The examples of high activity of the KARI described in that patent are those of Vibrio cholerae (DNA: sec.with ident.ident .: 389; protein, sec.with ident.no .: 390), Pseudomonas aeruginosa PA01, ( DNA: sec. With ident. No .: 422; protein, sec. With ident. No .: 423), and Pseudomonas fluorescens PF5 (DNA: sec. With ident. No .: 391; protein, sec. Ident No .: 392).

In addition, in U.S. Patent No. 20070092957 Al describes the construction of chimeric genes and the genetic manipulation of bacteria and yeast for the production of isobutanol by using the described biosynthetic routes.

Cultivation for production The yeast hosts or recombinant bacteria described in the present disclosure are cultured in fermentation media containing suitable carbon substrates. Suitable carbon substrates may include, but are not limited to, monosaccharides such as fructose, oligosaccharides such as lactose, maltose, galactose or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, and unpurified mixtures of renewable raw materials such as cheese whey permeate, corn steep liquor, beet molasses sugar bowl, and barley malt. Other carbon substrates may include ethanol, lactate, succinate or glycerol.

In addition, carbon substrates can also be single carbon substrates, such as carbon dioxide or ethanol, for which metabolic conversion in key biochemical intermediates has been demonstrated. In addition to the one and two carbon substrates, methylotrophic organisms are also known to use many other carbon-containing compounds, such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to use the carbon of methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993), 415-32, Editor (s): Murrell, J. Collin, Kelly, Don P. Editorial: Intercept, Andover, United Kingdom of Great Britain). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch Microbiol., 153: 485-489 (1990)). Therefore, it is contemplated that the carbon source employed in the present invention may encompass a wide variety of substrates that contain carbon and will only be limited by the choice of the organism.

Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, the preferred carbon substrates are glucose, fructose and sucrose, or mixtures of these with C5 sugars, such as xylose and / or arabinose, for yeast cells modified to use C5 sugars. Sucrose can be derived from renewable sugar sources, such as sugar cane, sugar beet, cassava, sweet sorghum, and mixtures of these. Glucose and dextrose can be derived from renewable grain sources through the saccharification of starch-based raw materials including grains, such as corn, wheat, rye, barley, oats and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through pretreatment and saccharification processes, as described, for example, in the co-pending United States joint patent application publication no. 2007 / 0031918A1, which is incorporated in the present description as a reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials that comprise cellulose and, optionally, also comprise hemicellulose, lignin, starch, oligosaccharides and / or monosaccharides. Biomass can also comprise other components, such as proteins and / or lipids. The biomass can be derived from a single source or can comprise a mixture derived from more than one source; for example, the biomass may comprise a mixture of corn cobs and corn stubble, or a mixture of grasses and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, papermaking sediments, organic waste, forestry and forestry residues. Examples of biomass include, but are not limited to, corn grains, corn cobs, crop residues, such as corn grain husks, corn stubbles, herbs, wheat, wheat straw, barley, barley straw. , hay, rice straw, panizo grass, scrap paper, sugarcane bagasse, sorghum, soybeans, components obtained from the grinding of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, manure, and mixtures of these.

In addition to the appropriate carbon source, the fermentation media must contain minerals, salts, cofactors, buffer solutions and other suitable components known to those skilled in the art, suitable for the growth of crops and the promotion of an enzymatic route comprising a bacterial DHAD [2Fe-2S]. Culture conditions Typically, the cells are cultured at a temperature in the range of about 20 ° C to about 40 ° C in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media, such as Luria Bertani broth (LB), Sabouraud Dextrose broth (SD), yeast medium broth (YM) or broth that includes a yeast nitrogen base , ammonium sulfate and dextrose (as the carbon / energy source) or YPD medium, a combination of peptone, yeast extract and dextrose in optimum proportions to cultivate the greatest number of strains of Saccharomyces cerevisiae. Other defined or synthetic growth media may also be used, and the means suitable for growth of the specific microorganism will be known to a person skilled in the art of microbiology or fermentation science. The use of known agents to modulate the repression of catabolites directly or indirectly, for example, cyclic adenosine 2 ': 3'-monophosphate, can also be incorporated into the fermentation medium.

The pH ranges suitable for yeast fermentation are typically from pH 3.0 to pH 9.0. , wherein a pH of 5.0 is preferred at a pH of 8.0 as an initial condition. Suitable pH ranges for the fermentation of other microorganisms are from a pH of 3.0 to a pH of 7.5, where a pH of 4.5.0 is preferred at a pH of 6.5 as an initial condition.

Fermentations can be carried out in aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

Continuous and discontinuous industrial fermentations Fermentation can be a discontinuous fermentation method. A classic discontinuous fermentation is a closed system in which the composition of the medium is defined at the beginning of the fermentation and is not subject to artificial alterations during fermentation. Therefore, at the beginning of the fermentation, the medium is inoculated with one or more desired organisms and the fermentation is allowed to take place without any addition to the system. However, typically, a "discontinuous" fermentation is discontinuous with respect to the addition of the carbon source and, frequently, attempts are made to control factors such as pH and oxygen concentration. In batch systems, the biomass and metabolite compositions of the system change constantly until the fermentation stops. Within the discontinuous cultures, the cells are moderated through a static delay phase until a logarithmic phase of high growth to end with a stationary phase, where the growth rate decreases or is interrupted. If they are not treated, the cells of the stationary phase will eventually die. The cells of the logarithmic phase are generally responsible for the mass production of the final or intermediate product.

A variation of the standard batch system is the discontinuous or batch feed system. Batch-fed fermentation processes are also suitable in the present invention and comprise a typical batch system, with the exception that the substrate is added in increments as the fermentation progresses. Batch feeding systems are useful when the repression of catabolites is apt to inhibit cell metabolism, and where it is desirable to have limited amounts of substrate in the media. Measurements of the actual substrate concentration in batch feeding systems is difficult and, therefore, calculated according to changes in measurable factors, such as pH, dissolved oxygen and partial pressure of waste gases, such as C02 Batch and discontinuous feed fermentations are common and well known in the art, and examples can be found in Thomas D. Brock in Biotechnology. A Textbook of Industrial Microbiology, second edition (1989) Sinauer Associates, Inc., Sunderland, MA. , or in Deshpande, Mukund V., Appl. Biochem. Biotechnol. , 36: 227, (1992), which are incorporated in the present description as a reference.

The fermentation culture can be adapted to continuous fermentation methods. Continuous fermentation is an open system, where a defined fermentation medium is continuously added to a bioreactor and extracted, simultaneously, an equal amount of conditioned medium for processing. Generally, continuous fermentation keeps the crops at a constant high density.

Continuous fermentation allows the modulation of a factor or any number of factors that affect cell growth or concentration of the final product. For example, a method will maintain a limiting nutrient, such as the nitrogen level or carbon source, at a fixed concentration and allow all other parameters to be moderated. In other systems, several factors that affect growth can be altered continuously, while the cell concentration, determined by the turbidity of the culture medium, remains constant. Continuous systems strive to maintain stable growth conditions and, thus, cell loss due to the medium that is extracted must be balanced against the rate of cell growth in the fermentation. Methods for modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the speed of product formation, are well known in the art of industrial microbiology and a variety of methods are detailed in Brock, supra. .

It is contemplated that discontinuous, discontinuous, continuous feed or any known fermentation modes are suitable for the growth of the described recombinant microbial host cell. Also I know it is contemplated that the cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for the production of isobutanol. Methods for isolating the product from the fermentation medium The biologically produced isobutanol can be isolated from the fermentation medium by the use of methods known in the art, such as those of ABE fermentations (see, for example, Durre, Appl Microbiol Biotechnol 49: 639-648 (1998), Groot et al., Process Biochem., 27: 61-75 (1992), "and references therein.) For example, solids can be extracted from the fermentation medium by centrifugation, filtration, decanting, or the like. be isolated from the fermentation medium by using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation or pervaporation.

Since isobutanol forms an azeotropic mixture with water and a low boiling point, distillation can be used to separate the mixture to its azeotropic composition. The distillation can be used in combination with another separation method to obtain separation around the azeotrope. Methods that can be used in combination with distillation to isolate and purify butanol include, but are not limited to, decanting, liquid-liquid extraction, adsorption and membrane-based techniques. In addition, butanol can be isolated by the use of azeotropic distillation when using a cosolvent (see, for example, Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so that the distillation can be used in combination with decantation to isolate and purify isobutanol. In this method, the fermentation broth containing isobutanol is distilled until approaching the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase can be returned to the first distillation column as reflux. The decanted organic phase rich in isobutanol can be further purified by distillation in a second distillation column.

In addition, isobutanol can also be isolated from the fermentation medium by the use of liquid-liquid extraction in combination with distillation. In this method isobutanol is extracted from the fermentation broth by using a liquid-liquid extraction with a suitable solvent. Then, the organic phase containing butanol is distilled to remove the butanol from the solvent.

Distillation in combination with adsorption can also be used to isolate isobutanol from the fermentation medium. In this method the fermentation broth containing the isobutanol is distilled until close to the composition azeotropic and then the rest of the water is extracted by the use of an adsorbent, such as molecular sieves (Aden et al.) Lignocellulosic Biomass to Ethanol Process Design and Economice Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, NREL Report / TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation can be used to isolate and purify isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled until approaching the azeotropic composition, and then the rest of the water is extracted by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245 , 199-210 (2004)).

EXAMPLES The present invention is defined in more detail through the following examples. It should be understood that while pointing to a preferred embodiment of the invention, these examples are illustrative only. From the above description and from these examples, a person skilled in the art will be able to establish the essential characteristics of this invention and, without departing from the spirit or scope thereof, may introduce various changes and modifications of the invention to adapt it to the various uses and conditions.

General methods The standard techniques of molecular cloning and recombinant DNA that are described in the examples are well known in the art and are described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, Y, (1989) (Maniatis) and in T. J. Silhavy, M. L. Bennan, and L. W. Enguist, Experiment with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984), and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987), and in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Suitable materials and methods for the maintenance and growth of bacterial cultures are well known in the art. Suitable techniques for use in the following examples can be found in the Manual of Methods for General Bacteriology (Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips , editors), American Society for Microbiology, Washington, DC. (1994)) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of cells Bacterial samples were obtained through Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified. shape.

The microbial strains were obtained from the collection of The American Type Culture Collection (ATCC), Manassas, VA, unless otherwise specified. The oligonucleotide primers used in the following examples were synthesized in Sigma-Genosys (Woodlands, TX) or Integrated DNA Technologies (Coralsville, IA).

The complete synthetic medium is described in Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

The analysis of the composition of the fermentation by-product is well known to those skilled in the art. For example, a high-performance liquid chromatography (HPLC) method uses a Shodex SH-1011 column with a Shodex SH-G column guard (both available from aters Corporation, Milford, A), with detection of refractive index (IR). Chromatographic separation is achieved by using 0.01 M H2SO4 as a mobile phase with a flow rate of 0.5 ml / min and a column temperature of 50 ° C. The retention time for isobutanol is approximately 47.6 minutes.

The meaning of the abbreviations is the following: "s" means second (s), "min" means minute (s), "h" means time (s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day (s), "μ ? " means microliter (s), "mi" means milliliter (s), "1" means liter, "mm" means millimeter (s), "mM" means millimolar, "μ?" means micromolar, "M" means molar, "mmol" means millimole (en), "μt ???" means micromol (s), "g" means gram (s), "pg" means microgram (s) and "ng" means nanogram (s), "PCR" stands for polymerase chain reaction, "OD" stands for optical density , "DO600" means the optical density measured at a wavelength of 600 nm, "kDa" means kilodaltons, "g" means constant of gravitation, "pb" means base pair (s), "pKb" means pair (es) ) of kilobases, "~" means approximately, "% w / v" means percent by weight / volume, "% v / v" means percent by volume / volume, "HPLC" means high performance liquid chromatography, and "CG" means gas chromatography.

Example 1 Identification of bacterial dihydroxy acid dehydratases with group [2Fe-2S] Phylogenetic analysis The phylogenetic relationships were determined by dihydroxy acid dehydratases (DHAD) and related proteins.

Related proteins were identified by searches of BlastP in publicly available databases through the use of amino acid sequences of the E. coli DHAD (sec. With ident. No .: 382), E. coli phosphogluconate dehydratase (EDD). , sec. with ident. no .: 384; coding region, sec. with ident. no .: 383); and arabicas dehydratase of Azospirillum brasiliense (sec. with ident. no .: 386; coding region, sec. with identification number: 385), with the following search parameters: Value of E = 10, word size = 3, Matrix = Blosum62, and opening of the interruption = 11 and extension of the interruption = 1. Blast searches employ the three different sequences of proteins generated by overlaying sets of sequence matches. The sequences were selected from the search results based on a cut-off value for E of 10"5 with the elimination of sequences with 95% identity, and sequences of a length of less than 350 amino acids and sequences of a length greater than 650 amino acids.The resulting set of 976 amino acid sequences included dihydroxy acid dehydratases, phosphogluconate dehydratases and aldonic acid dehydratases.

An HMM profile was generated from the experimentally verified DHADs described in Example 2. See below for details on the construction, calibration and search with this HM profile. A hmmer search using this HMM profile as the database query against the 976 sequences matched all the sequences with an E value of < 10"5. Multiple sequence alignments of the amino acid sequences were performed with the Clustal W algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc Acid Res. 22: 4673 4680) with the following parameters: 1) for parameters of alignment by pairs, an opening of the interruption = 10, extension of the interruption = 0.1, the matrix is Gonnet 250, and the mode - Slow-exact, 2) for parameters of multiple alignment, opening of The interruption = 10, extension of the interruption = 0.2, and the matrix is the Gonnet series.The phylogenetic trees were generated from sequence alignments based on the method of the nearest neighbor.A tree representing phylogenetic relationships among the 976 sequences are shown in Figure 2. Four main branches emerge from this analysis: "DHAD 4Fe-4S", "DHAD 2Fe-2S", "aldonic acid dehydratase" and "EDD" are marked, depending on the criteria used. they detail s forward A fifth small branch of 17 sequences is marked "Und" by "undefined".

The aligned sequences were initially analyzed to determine the presence of three cysteines that are essential for enzymatic activity and are probably involved in the coordination of Fe-S in the arabonato Azospirillum brasiliense dehydratase, which was reported as a group protein [4Fe-4S] (Watanabe, S et al., J. Biol. Chem. (2006) 281: 33521-33536). Each of the 976 sequences has the cysteines that correspond to two of the essential cysteines of arabonato dehydratase from Azospirillum brasiliense (at positions 124 and 197). Within the phylogenetic tree there is a branch of 168 sequences that includes the phosphogluconate dehydratase [4Fe-4S] of Zymomonas mobilis (Rodriguez, M. et al. (1996) Biochem Mol Biol Int. 38: 783-789). Only the four amino acids alanine, valine, or serine or glycine, but not cysteine, were found in the position of the third essential cysteine of the arabonato dehydratase of A. brasiliense (position 56). This branch of 168 sequences is marked in Figure 2 as "EDD".

A different branch of the tree contains 322 sequences, among which is the DHAD of group [4Fe-4S] known from E. coli. This branch of 322 sequences is marked in Figure 2 as "DHAD 4Fe-4S". All sequences within this branch, and in the 17-sequence branch ("Und"), contain glycine in the position corresponding to the third cysteine. The remaining 469 sequences, which are grouped into two branches and comprise both the aldonic acid recognizing the A. brasiliense arabido dehydratase and a set of DHAD possess the three cysteines. These cysteines are in positions 56, 129 and 201 in the DHAD of S. mutans (sec. With ident. No .: 168) and in positions 61, 135 and 207 in the DHAD of L. lactis (sec. with ident. no .: 232). In Figure 1 examples of regions of multiple sequence alignments including conserved cysteines are shown.

An additional analysis of multiple sequence alignments and phylogenetic trees was performed to identify specific (distinctive) residues of DHAD to distinguish DHAD from arabonato dehydratases and other dehydratases from aldonic acid. Among the sequences containing the three conserved cysteines specified, it was found that one of 274 sequences contained the DHADs of S. utans and L. lactis. The A. brasiliense arabido dehydratase was found in a separate group of 195 sequences. Multiple alignments of sequences containing the sequences of the group of 274 with the DHAD, the group of dehydratases of aldonic acid, and the branch of "DHAD [4Fe-4S]" were analyzed to determine the residues conserved in each position. A set of residues was detected that were preserved in most of both groups of DHAD, but not in the group of dehydratases of aldonic acid, and is shown in Table 4. In addition, residues that were conserved in the dehydratases were also found. of aldonic acid, but in neither of the two DHAD groups. These differentially conserved residues can act as determinants of substrate specificity in their respective enzymes.

Table 4. Residues conserved * with discrimination of DHAD of aldonic acid dehydratases Residue (s) retained in a majority of > 90% of representative cases.

* The numbering of the position is based on the position in the DHAD of S. mutans ** Not preserved The group of DHADs that forms the cyst of 274 sequences that does not include DHAD of group [4Fe-4S] of E. coli, phosphogluconate dehydratase of group [4Fe-4S] of Z. mobilis, or group arabonato dehydratase [4Fe- 4S) of A. brasiliense as reported was differentially identified from the other groups by phylogeny and conserved residues found in multiple sequence alignments, as described previously. In accordance with the proposal that the group includes DHAD with [2Fe-2S] groups, the DHAD of Arabidopsis thaliana and the DHAD of S. solfataricus are part of the group. Since Arabidopsis thaliana is a plant such as spinach, and DHAD from spinach has been identified as a DHAD with group [2Fe-2S] (Flint and Emptage (1988) J. Biol. Chem. 263: 3558 -3564), the DHAD of Arabidopsis thaliana can be a DHAD with a group [2Fe-2S]. It is described that the DHAD of S. solfataricus is resistant to oxygen as the DHAD of spinach having a [2Fe-2S] group (Kim and Lee (2006) J. Biochem. 139, 591-596), which is an indication that the DHAD of S. solfataricus can be a DHAD with a group [2Fe-2S].

The sequence of 274 sequences is marked in Figures 4A and 4B as "DHAD 2Fe-2S". 193 of these sequences are of bacterial origin. The three conserved cysteines and the conserved residues specified in Table 4 can be identified in multiple sequence alignments of the bacterial 193 DHADs by employing the alignment procedure described above. The sequences of the bacterial 193 DHAD [2Fe-2S] are given in the sequence listing, and the sequence identification numbers are listed in Table 2a.

Within the DHAD group [2Fe-2S], the identities of several bacterial proteins have been confirmed by functional analysis as DHAD, which is described in other examples of this invention. Other examples in the present invention show that the proteins of this group, such as the DHAD of S. mutans and the DHAD of L. lactis, contain a group [2Fe-2S].

Preparation of the HMM profile Seven bacterial DHADs that were identified as members of the phylogenetic group [2Fe-2S] were expressed in E. coli and dihydroxy acid dehydratase activity was tested as described in Example 2 below. These DHAD are from Nitrosomonas europaea (sec. With ident. No .: 310), Synechocystis sp. PCC6803 (sec. With ident. No .: 298), Streptococcus mutans (sec. With ident. No .: 168), Streptococcus ther ophilus (sec. With ident. No .: 164), Ralstonia metallidurans (sec. with ID number: 346), Ralstonia eutropha (sec. with ID: 344), and Lactococcus lactis (sec. with ID: 232). In addition, it was found that the DHAD of Flavobacterium johnsoniae (sec. With ident. No .: 230) had dihydroxy acid dehydratase activity when expressed in E. coli. The amino acid sequences of these experimentally determined functional bacterial DHADs were analyzed with the HMMER software package (the conceptual framework of the HMM profile is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis : probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235: 1501-1531), which follows the user's guide, which can be obtained from HMMER (Janelia Farm Research Campus, Ashburn, VA). The output of the HMMER software is a profile of hidden Markov models (HMM) that characterizes the input sequences. As mentioned in the user guide, the profile of the HMMs are statistical models of multiple sequence alignments. They capture information specific to a position regarding the degree of conservation of each column of the alignment and which amino acid residues are more likely to appear in each position. Thus, HMMs have a formal probabilistic base. The HMM profile for a large number of protein families is publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, VA).

The HMM profile was constructed as follows: Stage 1. Create a sequence alignment The eight sequences for the functionally verified DHADs listed above were aligned with Clustal W with the default parameters.

Stage 2. Create a HMM profile The hmmbuild program was executed on the set of sequences aligned with the predetermined parameters. The hmmbuild program reads the file from multiple sequence alignments, creates a new HMM profile, and saves this profile to a file. Using this program, an uncalibrated profile was generated from the multiple alignment for each of the sets of subunit sequences described above.

The following information based on the user guide of the HMMER program provides a description of how the hmmbuild program prepares an HMM profile. An HMM profile is able to model alignments with interruptions, for example, by including insertions and deletions, which allows the program to describe a complete conserved domain (instead of just a small uninterrupted reason). The insertions and deletions are modeled with the insertion (I) and suppression (D) states. All columns that contain more than a certain fraction x of characters of the interruption will be assigned as a column of inserts. The default value of x is 0.5. Each state of coincidence has an associated state I and a state D. The HMMER program invokes a group of three states (M / D / I) in the same consensus position in the alignment, a "node". These states are interconnected with arrows called probabilities of state transitions. The states M and I are emitters, while the states D are silent. The transitions are arranged so that, at each node, the M state is used (and a residue is aligned and written down) or the D state is used (and no residue is aligned, which produces a deletion character in the interruption, '-'). The insertions occur between nodes, and the I states have a self-transduction that allows one or more residues inserted between consensus columns to be produced.

The scores of the residuals in a matching state (ie emission scores of the matching state), or in an insertion state (ie, emission scores of the insertion state) are proportional to the Log_2 (p_x) / ( null_x). Where p_x is the probability of an amino acid residue, at a particular position of the alignment, according to the HMM profile, and null_x is the probability of conformity with the Null model. The Null model is a simple single-state probabilistic model with a pre-calculated set of emission probabilities for each of the 20 amino acids derived from the amino acid distribution in version 24 of SWISSPROT.

State transition scores are also calculated as logarithmic probability parameters and are proportional to Log_2 (t_x). Where t_x is the probability of transition to a sending or non-sending state.

Stage 3. Calibrate the HMM profile The HMM profile was read with hmmcalibrate (calibrate), which assigns a score to a large number of random sequences synthesized with the Profile (the predetermined amount of synthetic sequences). used is 5,000), adjusts an extreme value distribution (EVD) to the histogram of those scores and restores the HMM file that now includes the EVD parameters. These EVD parameters (μ and?) Are used to calculate the E values of normalized score ("bit scores") when the profile is searched in a database of protein sequences. The hmmcalibrate program writes two parameters in the HMM file on a line labeled "EVD"; These parameters are the parameters μ (location) and? (scale) of the extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of approximately the same length and composition of residues as SWISS-PROT. This calibration was performed once for the profile HMM The HMM profile calibrated for the set of DHAD sequences is provided in Table 1. The HMM profile is provided in a table that gives the probability of occurrence frequency of each amino acid at each position in the amino acid sequence. The highest probability is highlighted for each position. The first line for each position reports the emission scores of coincidences: the probability that each amino acid is in that state (the highest score is highlighted). The second line reports insertion emission scores, and the third line reports the state transitions scores: M? M, M? I, M? D; I? M, I? I; D? M, D? D; B? M; ME .

For example, the HMM profile for DHAD shows that methionine has a 1757 probability of being in the first position, the highest probability, which is highlighted. In the second position, glutamic acid has the highest probability, which is 1356. In the third position, the lysine has the highest probability, which is 1569.

Stage 4. Test the specificity and sensitivity of the HMM of the created profile The HMM profile was evaluated with hmmsearch (search), which reads an HMM profile from the hmmfile file (file) and searches for a sequence file with similar sequence matches. The searched sequence file contained 976 sequences (see above). During the search, the size of the database (parameter Z) was defined in one billion. This size configuration ensures that the significant E values against the current database will remain significant in the future foreseeable. The cutoff point for the value of E was defined at 10.

A hmmer search with the HMM profile generated from the alignment of the eight DHADs with experimentally verified function coincided with all 976 sequences with an E value of < 10"5. This result indicates that the members of the superfamily of the dehydratases share a significant sequence similarity, using a hmmer search with a cut-off point for the E value of 10" 5 to separate the dehydratases related to the DHADs. other proteins more remote, but related, as described above.

Example 2 Expression and characterization of bacterial dihydroxy acid dehydratases [2Fe-2S] in E. coli The ilvD coding regions of different bacteria, which from the phylogenetic analysis described in Example 1 are in the group [2Fe-2S], were expressed under the control of the T7 promoter in the vector pET28a (Novagen) in E. coli. Each coding region of ilvD was amplified with a specific forward primer with an Nhel restriction site and a specific reverse primer with a NotI restriction site (listed in Table 5).

Table 5. Secs. with no. of ident. of the primers used for PCR of regions coding for DHAD of the listed organisms.

The genomic DNA of each bacterial strain was used as a template. Genomic DNA was prepared from each strain listed in Table 1 with a MasterPure DNA purification kit (Epicenter, Madison, WI). The plasmid vector was amplified with the primers pET28 a-F (ot I) (sec.with ident.ident .: 397) and pET28a-R (Nhel) (sec. ident : 398) to eliminate the His tag region. The plasmid and gene fragments were digested with Nhel and Notl before ligation. The ligation mixture was transformed into competent (Top 10) cells (Invitrogen) from E. coli. Transformants were plated with LB-agar medium supplemented with 50 g / ml kanamycin. Positive clones that were confirmed by sequencing were transformed into the Tuner strain (DE3) (Novagen) of E. coli for expression. The selected colonies were cultured in liquid LB medium supplemented with kanamycin at 30 ° C. Induction was carried out by adding 0.5 mM of IPTG when the E. coli culture reached an OD of 0.3 to 0.4 at 600 nm. The culture was harvested after 5 hours of induction. The cell pellets were washed with Tris buffer solution (pH 8.0).

The enzymatic activity of the crude extract was analyzed at 37 ° C in the following manner. The cells to be analyzed for DHAD were suspended in 2-5 volumes of 50 mM Tris buffer solution and 10 mM MgSO4 (TM8), pH 8, and then broke by sonication at 0 ° C. The crude extract of the cell disruption was centrifuged to granulate the cell debris. The supernatants were removed and stored on ice until the time of the trial (the initial trial was performed within two hours of cell disruption). It was found that the DHADs analyzed here were stable in crude extracts kept on ice for a few hours. The activity was also maintained when small samples were frozen in liquid N2 and stored at -80 ° C.

The supernatants were analyzed with the reagent 2, 4-dinitropheni-1-hydrazine reagent, as described in Flint and Emptage (J. Biol. Chem. (1988) 263: 3558-64). When the activity was so high that it became necessary to dilute the crude extract to obtain an exact analysis, the dilution was done in 5 mg / ml of BSA in TM8.

Protein analyzes were performed with Pierce's Better Bradford reagent (cat # 23238) with BSA as standard. When necessary, dilutions for protein analyzes were made in TM8 buffer solution.

All DHADs were active when expressed in E. coli, and the specific activities are given in Table 6. The DHAD of Streptococcus mutans had the highest specific activity.

Table 6. Activity of bacterial DHAD [2Fe-2S] in E. coli Example 3 Purification and characterization of the DHAD of Streptococcus mutans expressed in E. coli The DHAD of S. mutans was characterized and purified Additionally . For the purification of DHAD from S. mutans, six liters of culture of the E. coli Tuner strain including plasmid pET28a with the ilvD of S. mutans were cultured and induced with IPTG. The enzyme was purified by cell disruption, as described in Example 2, in a 50 mM Tris buffer solution with a pH of 8.0 containing 10 mM MgCl2 (TM8 buffer solution), centrifuged to remove the debris of cells, and then the supernatant of the crude extract was loaded onto a Q Sepharose column (GE Healthcare) to elute the DHAD with an increased concentration of NaCl in the TM8 buffer solution. Fractions containing DHAD as a function of color appearance (brownish color due to the presence of the Fe-S group) were collected and loaded onto a Sephacryl S-100 column (GE Healthcare) to elute with TM8 buffer solution. As evaluated with the SDS gels, the purity of the protein eluted from the Sephacryl column was calculated at 60-80%. The activity of the partially purified enzyme was analyzed at 37 ° C as described by Flint et al. (J. Biol. Chem. (1988) 263 (8): 3558-64). The specific activity of the purified protein was 40 μ? T ??? min "1 mg" 1. It was calculated that the kca value for the purified enzyme was 50-70 s "1.

The stability in the presence of air of the purified DHAD was studied by incubating the purified enzyme at 23 ° C for several time intervals in the presence of ambient air followed by an activity analysis, as described above. The DHAD activity of Streptococcus mutans, as opposed to the DHAD similarly purified from E. coli, was stable even after 72 hours of incubation, as shown in Figures 4A and 4B, where Figure 4A shows the results of the DHAD of S. mutans and Figure 4B) shows the results of the DHAD of E. coli.

The visible UV spectrum of the purified S. mutans is shown in Figure 5. The amount of peaks above 300 nm is typical of proteins with [2Fe-2S] groups. The DHAD of S. mutans was reduced with sodium dithionite, and the EPR spectra were obtained at varying temperatures. Figure 6 shows the spectra measured at temperatures between -253 ° C (20 ° K) and -203 ° C (70 ° K). As the EPR spectrum of S. mutans can be measured up to 203 ° C (70 ° K), this is indicative that it contains a [2Fe-2S] group. It is well known, for example, that the EPR spectra of proteins containing groups [4Fe-4S] are not observable at temperatures much higher than 263 ° C (10 ° K). (See, for example, Rupp, et al., Biochimica et Biophysica Acta (1978) 537: 255-269.) Example 4 Construction of expression cassettes of dihydroxy acid dehydratase (DHAD) for Lactobacillus plantarum The purpose of this example is to describe how to clone and express a dihydroxy acid dehydratase gene (ilvD) of different bacterial origins in Lactobacillus plantarum PN0512 (ATCC PTA-7727). A pDMl transporter vector (sec. With ident number: 410) was used for the cloning and expression of ilvD genes of Lactococcus lactis, subspecies lactis NCD02118 (NCIMB 702118) [Godon et al., J. Bacteriol. (1992) 174: 6580-6589] and Streptococcus utans UA159 (ATCC 700610) in L. plantarum PN0512. Plasmid pDMl contains a minimum replicon pLF1 (-0.7 pKb) and toxin-antitoxin (TA) pemK-peml from plasmid pLFlATCC14917 from Lactobacillus plantarum, a P15A replicon from pACYC184, a marker of chloramphenicol for selection in E. coli and L. plantarum , and P30 synthetic promoter [Rud et al, Microbiology (2006) 152: 1011-1019]. Plasmid pLF1 (C.F. Lin et al., GenBank accession number: AF508808) is closely related to plasmid p256 [Sorvig et al., Microbiology (2005) 151: 421-431], whose number of copies was calculated in -5-10 copies per chromosome for L. plantarum NC7. A synthetic P30 promoter is derived from promoters for rRNA from L. plantarum that are known to be among the strongest promoters in lactic acid bacteria (LAB) [Rud et al. Microbiology (2005) 152: 1011-1019].

The ilvD coding region of Lactococcus lactis (SEQ ID NO: 231) was amplified by PCR from Lactococcus lactis genomic DNA, subspecies lactis, NCD02118, with 3T-ilvDLI (BamHI) primers (sec. ID number: 408) and 5B-ilvDLI (Notl) (sec. with ident. no .: 409). The genomic DNA of L. lactis, subspecies lactis, NCD02118, was prepared with a Puregene Gentra kit (QIAGEN, CA). The PCR product of 1.7 pKb from the ilvD of L. lactis,. { ilvDLI), was digested with Notl and treated with the Klenow fragment of the DNA polymerase to prepare blunt ends. The resulting ilvD coding region fragment of L. lactis was digested with BamHI and gel purified with the QIAGEN gel extraction kit (QIAGEN, CA). The pDMl plasmid was digested with ApaLI, treated with the Klenow fragment of the DNA polymerase to prepare blunt ends and then digested with BamHI. The ilvD coding region fragment of gel-purified L. lactis was ligated into the BamHI and ApaLI (blunt) sites of the pDMl plasmid. The ligation mixture was transformed into ToplO cells (Invitrogen, CA) from E. coli. The transformants were seeded for selection on plates with LB medium and chloramphenicol. Positive clones were selected by Sali digestion to give a fragment with an expected size of 5.3 pKb. Positive clones were additionally confirmed by DNA sequencing. The correct clone was named pDMl-ilvD (L. lactis).

The coding region of the ilvD of S. mutans UA159 (ATCC 700610) of the plasmid pET28a was cloned into the pDMl plasmid. The construction of pET28a containing the ilvD of S. mutans was described, for example, in Example 2. Plasmid pET28a containing the ilvD of S. mutans was digested with Xbal and Notl, treated with the Klenow fragment of the DNA polymerase to prepare blunt ends, and a 1.759 bp fragment containing the coding region of ilvD from S. mutans was gel purified. The pDMl plasmid was digested with BamHI, treated with the Klenow fragment of the DNA polymerase to prepare blunt ends, and then digested with PvuII. The gel-purified fragment containing the ilvD coding region of S. mutans was ligated into the BamHI (blunt) and PvuII sites of the pDMl plasmid. The ligation mixture was transformed into ToplO cells (Invitrogen, CA) from E. coli. The transformants were seeded for selection on plates with LB medium and chloramphenicol. Positive clones were selected by Clal digestion to give a fragment of an expected size of 5.5 pKb. The correct clone was named pD l-ilvD (S. mutans).

Example 5 Determination of DHAD activity expressed in L. plantarum PN0512 L. plantarum PN0512 was transformed with the plasmid pDMl-ilvD (L. lactis) or with pDMl-ilvD (S. mutans) by electroporation. The electrocompetent cells were prepared with the following procedure. Five ml of MRS medium for lactobacilli containing 1% glycine was inoculated with PN0512 cells and cultured overnight at 30 ° C. One hundred ml of MRS medium with 1% glycine was inoculated with the overnight culture at an OD600 = 0.1 and cultured at an OD600 = 0.7 at 30 ° C. Cells were harvested at 3700xg for 8 minutes at 4 ° C, washed with 100 ml cold of 1 mM MgCl2, centrifuged at 3700xg for 8 minutes at 4 ° C, washed with 100 ml cold PEG-1000 30% (81188, Sigma-Aldrich, St. Louis, MO), were recentrifuged at 3700xg for 20 minutes at 4 ° C and then resuspended in 1 ml cold 30% PEG-1000. Sixty μ? of electrocompetent cells were mixed with -100 ng of plasmid DNA in a cold electroporation cuvette 1 mm apart and electroporation was performed on a BioRad gene switch (Hercules, CA) at 1.7 kV, 25 pF and 400 O. cells were resuspended in 1 ml of MRS medium containing 500 mM sucrose and 100 mM MgCl 2, incubated at 30 ° C for two hours and then plated with MRS medium containing 10 g / ml chloramphenicol.

The transformants of L. plantarum PN0512 that include pDMl-ilvD (L. lactis) or pDMl-ilvD (S. mutans), as well as the control transformants with the pDMl vector alone, were grown overnight in RS medium for lactobacilli at 30 ° C. One hundred and twenty ml of MRS medium supplemented with 100 mM MOPS (pH 7.5), 40 μ? of ferric citrate, 0.5 mM of L-cysteine and 10 pg / ml of chloramphenicol were inoculated with the overnight culture at an OD600 = 0.1 in a 125 ml screw-cap flask for each sample overnight. The cultures were incubated anaerobically at 37 ° C until an OD600 of 1-2 was reached. The cultures were centrifuged at 3700xg for 10 minutes at 4 ° C. The granules were washed with 50 mM of potassium phosphate buffer solution with a pH of 6.2 (6.2 g / 1 of KH2P04 and 1.2 g / 1 of K2HP04) and recentrifuged. The granules were frozen and stored at -80 ° C until analyzed for DHAD activity. Samples of the cell extract were analyzed for DHAD activity with a dinitrophenylhydrazine-based method, as described in Example 2. The results of the DHAD activity are given in Table 7. The specific activity of the DHAD of L. lactis and the DHAD of S. mutans in L. plantarum PN0512 showed 0.02 and 0.06 pmol min "1 mg" 1, respectively, whereas the control sample of the vector did not show detectable activity.

Table 7. DHAD activity in L. plantarum PN0512.

Example 6 Expression of dihydroxy acid dehydratase from S. mutans in yeast The transporter vector pRS423 FBA ilvD (Strep) (sec.with ident.ID: 430) was used for the expression of DHAD from Streptococcus mutans. This transporter vector contained an origin of replication Fl (1423 to 1879) for maintenance in E. coli and an origin of 2 microns (nt 8082 to 9426) for replication in yeast. The vector has an FBA promoter (nt 2111 to 3108; sec. With ident. No .: 425) and an FBA terminator (nt 4861 to 5860). In addition, it carries the His marker (nt 504 to 1163) for selection in yeast and the ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The coding region of ilvD (nt 3116 to 4828) of Streptococcus mutans UA159 (ATCC 700610) is between the FBA promoter and the FBA terminator and forms a chimeric gene for expression.

To test the expression of Streptococcus DHAD mutans in yeast strain BY4741 (known in the art and obtainable from the ATCC, No. 201388), the pRS423 expression vector FBA IlvD (Strep) was transformed in combination with the empty vector pRS426 in BY4741 cells (which can obtained from the ATCC, no. 201388). The transformants were cultured in synthetic medium without histidine or uracil (Teknova). The culture in liquid medium for analysis was carried out by adding 5 ml of an overnight culture in 100 ml of medium in a 250 ml flask. The cultures were harvested when they reached an OD of 1 to 2 at 600 nm. The samples were washed with 10 ml of 20 mM Tris (pH 7.5) and then resuspended in 1 ml of the same Tris buffer solution. Samples were transferred to 2.0 ml tubes containing 0.1 mm silica (Lysing atrix B, MP biomedicals). Then, the mechanical breakdown of the cells was carried out in a macerator (BIO101). The supernatant was obtained by centrifugation in a microcentrifuge at 13,000 rpm at 4 ° C for 30 min. Typically, 0.06 to 0.1 mg of protein was used in the crude extract for the analysis of DHAD at 37 ° C, as described by Flint and Emptage (J. Biol. Chem. (1988) 263 (8): 3558-64). ) when using dinitrophenylhydrazine. The dehydratase of Streptococcus mutans had a specific activity of 0.24 pmol min "1 mg" 1 when expressed in yeast. A control strain containing empty vectors pRS423 and pRS426 had a history of activity in the range of 0.03 to 0. 06 μ ???? min "1 mg * 1.

Example 7 Expression of the IlvD gene of L. lactis in yeast The coding region of ilvD of L. lactis was amplified with the forward primer IlvD (Ll) -F (sec with ident. No .: 420) and reverse primer IlvD (Ll) -R (sec. With ident. No .: 421). The amplified fragment was cloned into the transport vector pNY13 by repair of the interruption. The vector pNY13 (sec. With ident.ID: 437) was derived from pRS423. This transporter vector contained an origin of replication Fl (1423 to 1879) for maintenance in E. coli and an origin of 2 microns (nt 7537 to 8881) for replication in yeast. The vector has an FBA promoter (nt 2111 to 3110) and an FBA terminator (nt 4316 to 5315). In addition, it carries the His marker (nt 504 to 1163) for selection in yeast and the ampicillin resistance marker (nt 6547 to 7404) for selection in E. coli.

Positive clones were selected on the basis of amplification with the forward and reverse primers for the coding region of ilvD and were further confirmed by sequencing. The new construct was designated pRS423 FBA ilvD (L. lactis). This construct was transformed into the yeast strain BY4743 (? LEU1) (Open Biosystems, Huntsville, AL; catalog number YSC1021-666629) together with the empty vector pRS426, as described in Example 6. Culture and analysis of yeast strains containing the expression vector were also carried out in accordance with the procedures described in Example 6. The dihydroxyacid dehydratase activity was determined in strains of yeast with the llvD gene of L. lactis was in the range of 0.05 to 0.17 ymol min "1 mg" 1. This activity was slightly above the control. Complementation experiments were carried out to investigate the expression of this DHAD and the DHADs of other bacteria (Example 8).

Example 8 Complementation between yeast strain with suppression of ILV3 and bacterial DHAD.

The endogenous DHAD enzyme of «S. Cerevisiae is encoded by the ILV3 gene and the protein is targeted to the mitochondria. The suppression of this gene results in the loss of endogenous DHAD activity and provides a test strain in which the expression of heterologous cytosolic DHAD activity can be easily evaluated. The suppression of ILV3 prevents the strain from growing in the absence of branched-chain amino acids. The expression of different bacterial DHAD was analyzed by determining its ability to complement the yeast strain with suppression of ILV3 so that the strain grows in the absence of branched chain amino acids.

Shuttle expression vectors containing ilvD gene sequences encoding the DHADs of the bacteria listed in Table 8 were constructed. The basic elements of these constructs The expression vectors were the same as those of the vector pRS423 FBA ilvD (strep) described in Example 6. Each of the coding regions of ilvD were prepared by PCR as described in Example 2 and cloned to replace the coding region of ilvD of Streptococcus ntutans in pRS423 FBA ilvD (Strep) and create the plasmids listed in Table 8. These expression constructs were transformed into the strain with ILV3 deletion, BY4741 ilv3:: URA3 which was prepared as follows. A cassette of disorder ilv3: URA3 was constructed with PCR amplification of the URA3 marker of pRS426 (ATCC No. 77107) with the primers "ILV3:: URA3 F" and "ILV3:: URA3 R", given as secs. with numbers Ident .: 431 and 432. These primers produced a 1.4 kb URA3 PCR product containing 5 'and 3' extensions of 70 bp identical to the sequences upstream and downstream of the chromosomal locus of ILV3 for homologous recombination. The PCR product was transformed into BY4741 cells (ATCC 201388) by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Y, pp. 201-202), and Resulting transformants were maintained in complete synthetic media without uracil and supplemented with 2% glucose at 30 ° C. The transformants were selected by PCR using the primers "ILV3 F Check" and "URA3 REV Check", given as secs. with numbers of identity: 433 and 434, to verify the integration in the correct site and the disruption of the endogenous locus of ILV3.

Transformants with the bacterial DHADs of Table 8 were selected on plates with synthetic medium for yeast without histidine. Then, the selected colonies were placed in patches on plates without valine, leucine or isoleucine. The strains containing the expression vectors listed in Table 8 were able to grow on these plates lacking branched chain amino acids, but this did not happen with the control strain that the control plasmid had. This result indicated that the DHADs of these bacteria were actively expressed in S. cerevisiae.

Table 8. Bacterial DHADs tested and expression vectors Origin organization of Sec. With no. of Designation the DHAD ident. of the acid sequence vector nuclei of the DHAD Nitrosomonas europaea 309 pRS423 FBA ATCC 19718 ilvD (europ) Synechocystis sp. PCC 297 pRS 23 FBA 6803 ilvD (Synech) Streptococcus 163 pRS423 FBA thermophilus L G 18311 ilvD (thermo) Ralstonia eutropha H16 406 pRS423 FBA (ATCC 17699) ilvD (H16) Lactococcus lactis 231 pRS423 FBA ilvD (L. lactis) Example 9 Purification and characterization of DHAD of Lactococcus lactis expressed in E. coli DHAD was purified and characterized from L. lactis. For the purification of the DHAD from L. lactis, 14 liters of culture of the Tuner strain (DE3) (Novagen) of E. coli, which harbors the plasmid pET28a containing the ilvD of L. lactis, were cultured and induced with IPTG . The enzyme was purified by cell disruption, as described in Example 2, in 120 ml of 50 mM Tris buffer solution with a pH of 8.0 containing 10 mM MgCl 2 (TM8 buffer solution), centrifuged to remove the cell residues and then the supernatant of the crude extract was loaded onto a 5 x 15 cm Q-Sepharose column (GE Healthcare) to elute the DHAD with an increased concentration of NaCl in the TM8 buffer solution. Fractions containing DHAD were collected, 1 M was prepared in and loaded onto a phenyl-Sepharose column of 2.6 x 15 cm, (GE Healthcare), equilibrated with 1 M of (H4) 2S04 in TM8 buffer solution and eluted with a decreasing gradient of (H4) 2S04. Fractions containing DHAD outside the phenyl-Sepharose column were collected and concentrated to 10 ml. This was loaded onto a 3.5 x 60 cm Superdex-200 column (GE Healthcare) and eluted with G? 8. The fractions containing DHAD activity were collected, concentrated and frozen as pellets in 2 (D. As determined with the SDS gels, the purity of the protein eluted from the Superdex-200 column was calculated to be> 80%. enzyme activity is analyzed at 37 ° C, as described by Flint et al. (J. Biol. Chem. (1988) 263 (8): 3558-64). The specific activity of the purified protein was 64 umol min "1 mg" 1 at a pH of 8 and 37 ° C. The kcat value for the purified enzyme was 71 s "1.

The stability in the presence of air of the purified DHAD was studied by incubating the purified enzyme at 23 ° C for several time intervals in the presence of ambient air followed by an activity analysis, as described above. The DHAD of L. lactis was almost fully active even after 20 hours of incubation in the presence of air, as shown in Figure 8.

The visible UV spectrum of purified L. lactis is shown in Figure 9. It is characteristic of proteins with [2Fe-2S] groups.

Example 10 Use of dihydroxy acid dehydratase to construct a route for the production of isobutanol in yeast The first three steps of a biosynthetic route of isobutanol are carried out with the enzymes acetolactate synthase, ketoacid reductoisomerase (KARI) and dihydroxy acid dehydratase. Acetolactate synthase is encoded by alsS. The KARI genes are known as ILV5 in yeast or ilvC in bacteria. When the -cetoisovalerate (KIV) is formed from pyruvate by the reaction of these three enzymes, it can be further converted to isobutanol in yeast with alcohol dehydrogenases.

The vector pLH532 (sec. With ident.ID .: 411) was constructed to express alsS and KARI genes. This vector is derived from the vector based on two pHR81 micras. In pLH532, the alsS coding region of B. subtilis (nt 14216 to 15931) was under the control of the CUP1 promoter (nt 15939 to 16386). There were two KARI genes in pLH532: the ilvC coding region of P. fluorescens Pf5 (nt 10192 to 11208) was under the control of the yeast ILV5 promoter (nt 11200 to 12390), and the ILV5 coding region of the yeast (nt 8118-9167) was placed under the control of the FBA promoter (nt 7454-8110). The selection marker was URA3 (numbers 3390 to 4190).

The yeast host for the production of isobutanol was BY4741 pdel:: FBAp-alsS-LEU2. This strain was constructed in the following manner. First, the expression plasmid pRS426-FBAp-alsS was constructed. The fragment of the 1.7 kb alsS coding region of pRS426:: GPD:: alsS:: CYC was isolated by gel purification followed by digestion of BbvCI and PacI. This plasmid has a chimeric gene containing the GPD promoter (sec. With ident.No .: 439), the alsS coding region of Bacillus subtilis (sec.with ident.No .: 438) and the CYC1 terminator (sec. with ID No.: 440), and described in Example 17 of U.S. Patent Publication no. 20070092957A1, which is incorporated herein by reference. The ILV5 fragment of plasmid pRS426:: FBA:: ILV5:: CYC, which is also described in Example 17 of U.S. Patent No. 20070092957, was removed by restriction digestion with BbvCI and Pací, and the remaining fragment of the 6.6 kb vector was gel purified. This vector has a chimeric gene containing the FBA promoter (sec. With ident.ID: 425) and CYC1 terminator which binds to the coding region of the ILV5 gene of S. cerevisiae (sec. 442). These two purified fragments were ligated overnight at 16 ° C and transformed into chemically competent TOP10 cells (Invitrogen) from E. coli. Transformants were obtained by plating cells on plates with LB / Ampl00 medium. The insertion of alsS in the vector was confirmed by restriction digestion pattern and PCR (primers N98SeqFl and N99SeqR2, secs with ident numbers: 412 and 413).

A pdcl · disruption cassette was created. FBAp-alSS-LEU2 by joining the FBAp-alsS segment of pRS426-FBAp-alsS to the LEU2 gene of pRS425 (ATCC, No. 77106) by SOE PCR (as described by Horton et al. (1989) Gene 77:61 -68) using the plasmid DNAs pRS426-FBAp-alsS and pRS425 as template, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, MA, catalog number F-540S) and primers 112590-48A and 112590-30B to D, given as sec. with no. ID: 414, secs. with numbers ID: 415, 416 and 417. The external primers for the SOE PCR (112590-48A and 112590-30D) contained 5 'and 3' 50 bp regions homologous to the upstream and downstream regions of the promoter and terminator. PDC1. The PCR fragment of the cassette finalized was transformed into BY4741 (ATCC No. 201388), and the transformants were maintained in complete synthetic media without leucine and supplemented with 2% glucose at 30 ° C by the use of standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Y, pp. 201-202). Transformants were selected by PCR using the primers 112590-30E and 112590-30F, given as secs. with numbers Ident .: 419 and 418, to verify the integration in the PDCl locus with suppression of the coding region of PDCl. The correct transformants have the genotype: BY4741 pdcl:: FBAp-alsS-LEU2.

To test whether the DHAD of Streptococcus mutans encoding the ilvD could be used for butanol biosynthesis, the expression vector containing this ilvD, pRS423 FBA ilvD (strep), prepared in Example 6, was cotransformed with the vector pLH532 into a yeast strain BY4741 pdcl:.-FBAp-alsS-LEU2. The preparation, transformation and the growth medium of competent cells for the selection of the transformants were the same as those described in Example 6. The selected colonies were cultured under oxygen limiting conditions in 15 ml of medium in 20 ml serum bottles with stoppers . The bottles were incubated at 30 ° C on a shaker with a constant speed of 225 rotations per minute. After 48 hours of incubation, the samples were analyzed with HPLC to determine the presence of isobutanol. The result of the HPLC analysis is shown in Figure 7. The presence of isobutanol was indicated by a peak with a retention time of 47,533 minutes. This result demonstrated that the ilvD gene expression of Streptococcus mutans, together with the expression of the alsS and KARI genes, led to the production of isobutanol in yeast.

Example 11 Consultation of updated databases to identify other dihydroxy acid dehydratases [2Fe-2S] Later a second query was made in the updated public database to discover new sequences of dihydroxy acid dehydratases [2Fe-2S]. At the 95% identity cut-off point, an initial set of 1425 sequences was generated from a database query, as described in Example 1, "Phylogenetic Analysis". Multiple sequence alignments were then run with ClustalW, as described in Example 1. The sequences were subsequently analyzed to determine the following residues conserved at the corresponding positions in the DHAD of S. mutans: cysteines at positions 56, 129 and 201, aspartic acid in position 88, arginine or asparagine in position 142, asparagine in position 208, and leucine in position 454. In an addition to the original set of 193, 88 novel dihydroxy acid dehydratases [2Fe-2S] were identified of bacteria and are listed in Table 2b.

Table 1 HMMER2.0 [2.2 g] Name and version of the program ÑAME dhad_for_hmm Name of the input sequence alignment file LENG 564 Length of alignment: includes insertions / deletions ALPH Amino Type of waste MAP yes (yes) Map of the matching states for alignment columns COM / app / public / hmmer / currentibin / hmmbuild (build) Commands used to generate the file: this means that hmmbuild -F dhad-exp_hmm dhadjor hmm was applied. aln (default parameters) to the alignment file COM / app / public / hmmer / current / bin / Commands used to generate the file: this means that hmmcalibrate hmmcalibrate (calibrate) dhad-expjimm (default parameters) was applied to the profile hmm NSEQ 8 Number of sequences in the alignment file DATE: Tuesday, June 3, 10:48:24, 2008 File generation date XT -8455 -4 -1000 -1000-8455 -4 -8455 -4 The distribution of transition probabilities for the null model (only G state) NULT -4 -8455 The distribution of probability of emission of symbols for the null model ( G state); NULE 595 -1558 85338 -294453 -1158 197249902 - consists of integers K (for example, 4 or 20). The probability of null values used for 1085 -142 -21 -31345531 201 384 -199 to convert these again into model probabilities is 1 / K.

EVD -499.6509700.086142 The distribution parameters of extreme values, μ and lambda, respectively; both floating point values. Lambda is positive and nonzero. These values are defined when the model is calibrated with hmmcalibrate.

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Claims (14)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method to identify DHAD enzymes [2Fe-2S], characterized in that it comprises: a) search in a database one or more amino acid sequences with a profile of hidden Markov models prepared using the sec proteins. with no. ID: 164, 168, 230, 232, 298, 310, 344 and 346, wherein a match with an E value less than 10"5 provides a first subset of sequences, whereby the first subset of sequences corresponds to one or more proteins related to DHAD; b) analyzing the first subset of sequences corresponding to one or more DHAD-related proteins of step (a) to determine the presence of three conserved cysteines corresponding to positions 56, 129 and 201 in the amino acid sequence of the dihydroxy acid dehydratase (sec .: Ident .: 168) of Streptococcus mutans, by virtue of which a second subset of sequences encoding DHAD enzymes [2Fe-2S] is identified; Y c) analyze the second subset of sequences of step (b) to determine the presence of distinctive conserved amino acids at positions corresponding to positions in the amino acid sequence of the DHAD of Streptococcus mutans (SEQ ID NO: 168) which are aspartic acid in the position 88, arginine or asparagine at position 142, asparagine at position 208, and leucine at position 454, whereby a third subset of sequences encoding DHAD enzymes [2Fe-2S] is also identified.

2. The method in accordance with the claim 1, characterized in that it also comprises: d) expressing a polypeptide having a sequence identifiable by any or all of steps a), b) and e) in a cell; Y e) confirm that polypeptide has activity DHAD in the cell.

3. The method according to claim 1, characterized in that it also comprises: d) purifying a protein encoded by an identifiable sequence by any or all of steps a), b) and c); Y e) confirm that the protein is a DHAD enzyme [2Fe-2S] by visible ultraviolet and EPR spectroscopy.

4. The method according to claim 1, characterized in that it comprises selecting one or more sequences corresponding to sequences of bacterial DHAD [2Fe-2S] enzymes identified in any or all of steps a), b) and e).

5. The method according to claim 2, characterized in that the cell lacks endogenous DHAD activity.

6. The method according to claim 4, characterized in that it also comprises: d) expressing one or more of the selected sequences corresponding to the enzyme sequences DHAD [2Fe-2S] bacterial in a cell; Y e) confirm that the enzyme sequence has DHAD activity in the cell.

7. The method according to claim 4, characterized in that it also comprises: d) purifying a protein encoded by one or more of the selected sequences corresponding to the sequences of bacterial DHAD [2Fe-2S] enzymes, by virtue of which a purified protein is produced; Y e) confirm that the protein is a DHAD enzyme [2Fe-2S] by visible ultraviolet and EPR spectroscopy.

8. A microbial host cell characterized in that it comprises at least one heterologous DHAD [2Fe-2S] enzyme identifiable by the method according to any of claims 1-7.

9. The microbial host cell is non-conformational with claim 8, characterized in that the cell is a bacterial cell or a yeast cell.

10. The microbial host cell according to claim 9, characterized in that the bacterial host cell is a member of a genus of bacteria selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus , Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevijbacteriu-n, Lactococcus, Leuconostoc, Oenococcus, Pediococcus and Streptococcus.

11. The microbial host cell according to claim 9, characterized in that the yeast cell is a member of a yeast genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

12. The microbial host cell according to claim 8, characterized in that the cell produces isobutanol.

13. A method for the production of isobutanol characterized in that it comprises: a) providing the microbial host cell according to claim 8, wherein the host cell comprises a biosynthetic route of isobutanol; Y b) culturing the host cell of step (a) under conditions in which isobutanol is produced.

14. A method for the conversion of 2,3-dihydroxyisovalerate to -cetoisovalerate characterized in that it comprises: a) providing the microbial host according to claim 8 and a source of 2,3-dihydroxyisovalerate; Y b) culturing the microbial host cell of (a) under conditions in which 2, 3-dihydroxyisovalerate is converted to α-ketoisovalerate.